Engineered proteins

ABSTRACT

Provided herein are engineered proteins. In some embodiments, the engineered proteins comprise a RuvC DNA cleavage domain comprising one or more modifications, relative to a naturally occurring domain. In some embodiments, the engineered proteins comprise a chimeric domain, for example a chimeric helical I domain. In some embodiments, the engineered proteins are chimeric proteins, comprising at least two domains, each domain derived from a different source, for example derived from SEQ ID NO: 1 and SEQ ID NO: 2. Also provided are systems comprising these engineered proteins, and methods of making and using said engineered proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent Application No. PCT/US2020/036505, filed on Jun. 5, 2020, which claims priority to U.S. Provisional Patent Application Nos. 62/858,750, filed on Jun. 7, 2019, 62/944,892, filed on Dec. 6, 2019 and 63/030,838, filed on May 27, 2020, the contents of each of which are incorporated herein by reference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 4, 2021 is named SCRB_011_04US_SeqList_ST25 and is 3.63 MB in size.

BACKGROUND

The CRISPR-Cas systems confer bacteria and archaea with acquired immunity against phage and viruses. Intensive research over the past decade has uncovered the biochemistry of these systems. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets. Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.

To date, only a few Class 2 CRISPR/Cas systems have been discovered that have been widely used. Thus, there is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations) that have been optimized and/or offer improvements over earlier generation systems for utilization in a variety of therapeutic, diagnostic, and research applications.

SUMMARY

In some aspects, the present disclosure provides variants of a reference CasX nuclease protein, wherein the CasX variant is capable of forming a complex with a guide nucleic acid (NA), and wherein the complex can bind a target DNA, wherein the target DNA comprises non-target strand and a target strand, and wherein the CasX variant comprises at least one modification relative to a domain of the reference CasX and exhibits one or more improved characteristics as compared to the reference CasX protein. The domains of the reference CasX protein include: (a) a non-target strand binding (NTSB) domain that binds to the non-target strand of DNA, wherein the NTSB domain comprises a four-stranded beta sheet; (b) a target strand loading (TSL) domain that places the target DNA in a cleavage site of the CasX variant, the TSL domain comprising three positively charged amino acids, wherein the three positively charged amino acids bind to the target strand of DNA, (c) a helical I domain that interacts with both the target DNA and a spacer region of a guide NA, wherein the helical I domain comprises one or more alpha helices; (d) a helical II domain that interacts with both the target DNA and a scaffold stem of the guide NA; (e) an oligonucleotide binding domain (OBD) that binds a triplex region of the guide NA; and (f) a RuvC DNA cleavage domain.

In some aspects, the present disclosure provides variants of a reference guide nucleic acid (gNA) capable of binding a CasX protein, wherein the reference guide nucleic acid comprises at least one modification in a region compared to the reference guide nucleic acid sequence, and the variant exhibits one or more improved characteristics compared to the reference guide RNA. The regions of the scaffold of the gNA include: (a) an extended stem loop; (b) a scaffold stem loop; (c) a triplex; and (d) pseudoknot. In some cases, the scaffold stem of the variant gNA further comprises a bubble. In other cases, the scaffold of the variant gNA further comprises a triplex loop region. In other cases, the scaffold of the variant gNA further comprises a 5 unstructured region.

In some aspects, the present disclosure provides gene editing pairs comprising the CasX proteins and gNAs of any of the embodiments described herein.

In some aspects, the present disclosure provides polynucleotides and vectors encoding the CasX proteins, gNAs and gene editing pairs described herein. In some embodiments, the vectors are viral vectors such as an Adeno-Associated Viral (AAV) vector or a lentiviral vector. In other embodiments, the vectors are non-viral particles such as virus-like particles or nanoparticles.

In some aspects, the present disclosure provides cells comprising the polynucleotides, vectors, CasX proteins, gNAs and gene editing pairs described herein. In other aspects, the present disclosure provides cells comprising target DNA edited by the methods of editing embodiments described herein.

In some aspects, the present disclosure provides kits comprising the polynucleotides, vectors, CasX proteins, gNAs and gene editing pairs described herein.

In some aspects, the present disclosure provides methods of editing a target DNA, comprising contacting the target DNA with one or more of the gene editing pairs described herein, wherein the contacting results in editing of the target DNA.

In other aspects, the disclosure provides methods of treatment of a subject in need thereof, comprising administration of the gene editing pairs or vectors comprising or encoding the gene editing pairs of any of the embodiments described herein.

In another aspect, provided herein are gene editing pairs, compositions comprising gene editing pairs, or vectors comprising or encoding gene editing pairs, for use as a medicament.

In another aspect, provided herein are gene editing pairs, compositions comprising gene editing pairs, or vectors comprising or encoding gene editing pairs, for use in a method of treatment, wherein the method comprises editing or modifying a target DNA; optionally wherein the editing occurs in a subject having a mutation in an allele of a gene wherein the mutation causes a disease or disorder in the subject, preferably wherein the editing changes the mutation to a wild type allele of the gene or knocks down or knocks out an allele of a gene causing a disease or disorder in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 is a diagram showing an exemplary method of making CasX protein and guide RNA variants of the disclosure using Deep Mutational Evolution (DME). In some exemplary embodiments, DME builds and tests nearly every possible mutation, insertion and deletion in a biomolecule and combinations/multiples thereof, and provides a near comprehensive and unbiased assessment of the fitness landscape of a biomolecule and paths in sequence space towards desired outcomes. As described herein, DME can be applied to both CasX protein and guide RNA.

FIG. 2 is a diagram and an example fluorescence activated cell sorting (FACS) plot illustrating an exemplary method for assaying the effectiveness of a reference CasX protein or single guide RNA (sgRNA), or variants thereof. A reporter (e.g. GFP reporter) coupled to a gRNA target sequence, complementary to the gRNA spacer, is integrated into a reporter cell line. Cells are transformed or transfected with a CasX protein and/or sgNA variant, with the spacer motif of the sgRNA complementary to and targeting the gRNA target sequence of the reporter. Ability of the CasX:sgRNA ribonucleoprotein complex to cleave the target sequence is assayed by FACS. Cells that lose reporter expression indicate occurrence of CasX:sgRNA ribonucleoprotein complex-mediated cleavage and indel formation.

FIG. 3A and FIG. 3B are heat maps showing the results of an exemplary DME mutagenesis of the reference sgRNA encoded by SEQ ID NO: 5, as described in Example 3. FIG. 3A shows the effect of single base pair (single base) substitutions, double base pair (double base) substitutions, single base pair insertions, single base pair deletions, and a single base pair deletion plus at single base pair substitution at each position of the reference sgRNA shown at top. FIG. 3B shows the effect of double base pair insertions and a single base pair insertion plus a single base pair substitution at each position of the improved reference sgRNA. The reference sgRNA sequence of SEQ ID NO: 5 is shown at the top of FIG. 3A and bottom of FIG. 3B. In FIG. 3A and FIG. B, Log₂ fold enrichment of the variant in the DME library relative to the reference sgRNA following selection is indicated in grayscale. Enrichment is a proxy for activity, where greater enrichment is a more active molecule. The results show regions of the reference sgRNA that should not be mutated and key regions that are targeted for mutagenesis.

FIG. 4A shows the results of exemplary DME experiments using a reference sgRNA, as described in Example 3. The improved reference sgNA (an sgRNA) with a sequence of SEQ ID NO: 5 is shown at top, and Log₂ fold enrichment of the variant in the DME library relative to the reference sgRNA following selection is indicated in grayscale. Enrichment is a proxy for activity, where greater enrichment is a more active molecule. The heat map shows an exemplary DME experiment showing four replicates of a library where every base pair in the reference sgRNA has been substituted with every possible alternative base pair.

FIG. 4B is a series of 8 plots that compare biological replicates of different DME libraries. The Log₂ fold enrichment of individual variants relative to the reference sgRNA sequence for pairs of DME replicates are plotted against each other. Shown are plots for single deletion, single insertion and single substitution DME experiments, as well as wild type controls, and the plots indicate that there is a good amount of agreement for each replicate.

FIG. 4C is a heat map of an exemplary DME experiment showing four replicates of a library where every location in the reference sgRNA has undergone a single base pair insertion. The DME experiment used a reference sgRNA of SEQ ID NO: 5 (at top), and was performed as described in Example 3. Log₂ fold enrichment of the variant in the DME library relative to the reference sgRNA following selection is indicated in grayscale.

FIGS. 5A-5E are a series of plots showing that sgNA variants can improve gene editing by greater than two fold in an EGFP disruption assay, as described in Examples 2 and 3. Editing was measured by indel formation and GFP disruption in HEK293 cells carrying a GFP reporter. FIG. 5A shows the fold change in editing efficiency of a CasX sgRNA reference of SEQ ID NO: 4 and a variant of the reference which has a sequence of SEQ ID NO: 5, across 10 targets. When averaged across 10 targets, the editing efficiency of sgRNA SEQ ID NO: 5 improved 176% compared to SEQ ID NO: 4. FIG. 5B shows that further improvement of the sgRNA scaffold of SEQ ID NO: 5 is possible by swapping the extended stem loop sequence for additional sequences to generate the scaffolds whose sequences are shown in Table 2. Fold change in editing efficiency is shown on the Y-axis. FIG. 5C is a plot showing the fold improvement of sgNA variants (including a variant with SEQ ID NO: 17) generated by DME mutations normalized to SEQ ID NO: 5 as the CasX reference sgRNA. FIG. 5D is a plot showing the fold improvement of sgNA variants of sequences listed in Table 2, which were generated by appending ribozyme sequences to the reference sgRNA sequence, normalized to SEQ ID NO: 5 as the CasX reference sgRNA. FIG. 5E is a plot showing the fold improvement normalized to the SEQ ID NO: 5 reference sgRNA of variants created by both combining (stacking) scaffold stem mutations showing improved cleavage, DME mutations showing improved cleavage, and using ribozyme appendages showing improved cleavage. The resulting sgNA variants yield 2 fold or greater improvement in cleavage compared to SEQ ID NO: 5 in this assay. EGFP editing assays were performed with spacer target sequences of E6 and E7.

FIG. 6 shows a Hepatitis Delta Virus (HDV) genomic ribozyme used in exemplary gNA variants (SEQ ID NOs: 18-22).

FIGS. 7A-7I are a series of heat maps showing the effect of single amino acid substitutions, single amino acid insertions, and deletions at each amino acid position in a reference CasX protein of SEQ ID NO: 2, as described in Example 4. Data were generated by a DME assay run at 37° C. The Y-axis shows each possible substitution or insertion (from top to bottom: R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y or V; boxes indicate the amino acid identity of the reference protein), the X-axis shows the amino acid position in the reference CasX protein. Log₂ fold enrichment of the CasX variant protein relative to the reference CasX protein of SEQ ID NO: 2 in a DME library following enrichment is indicated. As used herein, “enrichment” is a proxy for activity, where greater enrichment is a more active molecule. (*)s indicate active sites. FIGS. 7A-7D show the effect of single amino acid substitutions. FIGS. 7E-7H show the effect of single amino acid insertions. FIG. 7I shows the effect of single amino acid deletions.

FIGS. 8A-8C are a series of heat maps showing the effect of single amino acid substitutions, single amino acid insertions and deletions at each amino acid position in a reference CasX protein of SEQ ID NO: 2, as described in Example 4. Data were generated by a DME assay run at 45° C. FIG. 8A shows the effect of single amino acid substitutions. FIG. 8B shows the effect of single amino acid insertions. FIG. 8C shows the effect of single amino acid deletions. For all of FIGS. 8A-8C, The Y-axis shows each possible substitution or insertion (from top to bottom: R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y or V; boxes indicate the amino acid identity of the reference protein), the X-axis shows the amino acid position in the reference CasX protein. Log₂ fold enrichment of the CasX variant protein relative to the reference CasX protein of SEQ ID NO: 2 in a DME library following enrichment is indicated in grayscale, where greater enrichment is a more active molecule. (*)s indicate active sites. Running this assay at 45° C. enriches for different variants than running the same assay at 37° C. (see FIGS. 7A-7I), thereby indicating which amino acid residues and changes are important for thermostability and folding.

FIG. 9 shows a survey of the comprehensive mutational landscape of all single mutations of a reference CasX protein of SEQ ID NO: 2. On the Y-axis, fold enrichment of CasX variants relative to the reference CasX protein for single substitutions (top), single insertions (middle) or single deletions (bottom). On the X-axis, amino acid position in the reference CasX protein. Key regions that yield improved CasX variants are the initial helix region and regions in the RuvC domain bordering the target strand loading (TLS) domain, as well as others.

FIG. 10 is a plot showing that the evaluated CasX variant proteins improved editing greater than three-fold relative to a reference CasX protein in the EGFP disruption assay, as described in Example 5. CasX proteins were tested for their ability to cleave an EGFP reporter at 2 different target sites in human HEK293 cells, and the normalized improvement in genome editing at these sites over the basic reference CasX protein of SEQ ID NO: 2 is shown. Variants, from left to right (indicated by the amino acid substitution, insertion or deletion at the given residue number) are: Y789T, [P793], Y789D, T72S, I546V, E552A, A636D, F536S, A708K, Y797L, L792G, A739V, G791M, {circumflex over ( )}G661, A788W, K390R, A751S, E385A, {circumflex over ( )}P696, {circumflex over ( )}M773, G695H, {circumflex over ( )}AS793, {circumflex over ( )}AS795, C477R, C477K, C479A, C479L, I55F, K210R, C233S, D231N, Q338E, Q338R, L379R, K390R, L481Q, F495S, D600N, T886K, A739V, K460N, I199F, G492P, T153I, R591I, {circumflex over ( )}AS795, {circumflex over ( )}AS796,889, E121D, S270W, E712Q, K942Q, E552K, K25Q, N47D, {circumflex over ( )}T696, L685I, N880D, Q102R, M734K, A724S, T704K, P224K, K25R, M29E, H152D, S219R, E475K, G226R, A377K, E480K, K416E, H164R, K767R, I7F, M29R, H435R, E385Q, E385K, I279F, D489S, D732N, A739T, W885R, E53K, A238T, P283Q, E292K, Q628E, R388Q, G791M, L792K, L792E, M779N, G27D, K955R, S867R, R693I, F189Y, V635M, F399L, E498K, E386S, V254G, P793S, K188E, QT945KI, T620P, T946P, TT949PP, N952T, K682E, K975R, L212P, E292R, I303K, C349E, E385P, E386N, D387K, L404K, E466H, C477Q, C477H, C479A, D659H, T806V, K808S, {circumflex over ( )}AS797, V959M, K975Q, W974G, A708Q, V711K, D733T, L742W, V747K, F755M, M771A, M771Q, W782Q, G791F, L792D, L792K, P793Q, P793G, Q804A, Y966N, Y723N, Y857R, S890R, S932M, L897M, R624G, S603G, N737S, L307K, I658V {circumflex over ( )}PT688, {circumflex over ( )}SA794, S877R, N580T, V335G, T620S, W345G, T280S, L406P, A612D, A751 IS, E386R, V351M, K210N, D40A, E773G, H207L, T62A, T287P, T832A, A893S, {circumflex over ( )}V14, {circumflex over ( )}AG13, R11V, R12N, R13H, {circumflex over ( )}Y13, R12L, {circumflex over ( )}Q13, V15S, {circumflex over ( )}D17. {circumflex over ( )} indicate insertions, [ ] indicate deletions.

FIG. 11 is a plot showing individual beneficial mutations can be combined (sometimes referred to as “stacked”) for even greater improvements in gene editing activity. CasX proteins were tested for their ability to cleave at 2 different target sites in human HEK293 cells using the E6 and E7 spacers targeting an EGFP reporter, as described in Example 5. The variants, from left to right, are: S794R+Y797L, K416E+A708K, A708K+[P793], [P793]+P793AS, Q367K+I425S, A708K+[P793]+A793V, Q338R+A339E, Q338R+A339K, S507G G508R, L379R+A708K+[P793], C477K+A708K+[P793], L379R+C477K+A708K+[P793], L379R+A708K+[P793]+A739V, C477K+A708K+[P793]+A739V, L379R+C477K+A708K+[P793]+A739V, L379R+A708K+[P793]+M779N, L379R+A708K+IP793I+M771N, L379R+A708K+[P793]+D489S, L379R+A708K+[P793]+A739T, L379R+A708K+[P793]+D732N, L379R+A708K+[P793]+G791M, L379R+A708K+[P793]+Y797L, L379R+C477K+A708K+[P793]+M779N, L379R+C477K+A708K+[P793]+M771N, L379R+C477K+A708K+[P793]+D489S, L379R+C477K+A708K+[P793]+A739T, L379R+C477K+A708K+[P793]+D732N, L379R+C477K+A708K+[P793]+G791M, L379R+C477K+A708K+[P793]+Y797L, L379R+C477K+A708K+[P793]+T620P, A708K+[P793]+E386S, E386R+F399L+[P793] and R4581I+A739V of the reference CasX protein of SEQ ID NO: 2. [ ] refer to deleted amino acid residues at the specified position of SEQ ID NO: 2.

FIG. 12A and FIG. 12B are a pair of plots showing that CasX protein and sgNA variants when combined, can improve activity more than 6-fold relative to a reference sgRNA and reference CasX protein pair. sgNA:protein pairs were assayed for their ability to cleave a GFP reporter in HEK293 cells, as described in Example 5. On the Y-axis, the fraction of cells in which expression of the GFP reporter was disrupted by CasX mediated gene editing are shown. FIG. 12A shows CasX protein and sgNAs that were assayed with the E6 spacer targeting GFP. FIG. 12B shows CasX protein and sgNAs that were assayed with the E7 spacer targeting GFP. iGFP stands for “inducible GFP.”

FIG. 13A, FIG. 13B and FIG. 13C show that making and screening DME libraries has allowed for generation and identification of variants that exhibit a 1 to 81-fold improvement in editing efficiency, as described in Examples 1 and 3. FIG. 13A shows an RFP+ and GFP+ reporter in E. coli cells assayed for CRISPR interference repression of GFP with a reference nuclease dead CasX protein and sgNA. FIG. 13B shows the same reporter cells assayed for GFP repression with nuclease dead CasX variants screened from a DME library. FIG. 13C shows improved editing efficiency of a selected CasX protein and sgNA variant compared to the reference with 5 spacers targeting the endogenous B2M locus in HEK 293 human cells. The Y axis shows disruption in B2M staining by HLA1 antibody indicating gene disruption via CasX editing and indel formation. The improved CasX variants improved editing of this locus up to 81-fold over the reference in the case of guide spacer #43. CasX pairs with the reference sgRNA:protein pair of SEQ ID NO: 5 and SEQ ID NO: 2, and CasX variant protein of L379R+A708K+[P793] of SEQ ID NO: 2, assayed with the sgNA variant with a truncated stem loop and a T10C substitution, which is encoded by a sequence of TACTGGCGCCTTTATCTCATTACTTGAGAGCCATCACCAGCGACTATGTCGTATGG GTAAAGCGCTTACGGACTTCGGTCCGTAAGAAGCATCAAAG (SEQ ID NO: 23), are indicated. The following spacer sequences were used: #9: GTGTAGTACAAGAGATAGAA (SEQ ID NO: 24); #14: TGAAGCTGACAGCATTCGGG (SEQ ID NO: 25), #20: tagATCGAGACATGTAAGCA (SEQ ID NO: 26); #37: GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 27) and #43: AGGCCAGAAAGAGAGAGTAG (SEQ ID NO: 28).

FIGS. 14A-14F are a series of structural models of a prototypic CasX protein showing the location of mutations in CasX variant proteins of the disclosure which exhibit improved activity. FIG. 14A shows a deletion of P at 793 of SEQ ID NO: 2, with a deletion in a loop that may affect folding. FIG. 14B shows a replacement of Alanine (A) by Lysine (K) at position 708 of SEQ ID NO: 2. This mutation is facing the gNA 5′ end plus a salt bridge to the gNA. FIG. 14C shows a replacement of Cysteine (C) by Lysine (K) at position 477 of SEQ ID NO: 2. This mutation is facing the gNA. There is salt bridge to the gNAbb (gNA phosphase backbone) at approximately base 14 that may be affected. This mutation removes a surface exposed cysteine.

FIG. 14D shows a replacement of Leucine (L) with Arginine (R) at position 379 of SEQ ID NO: 2. There is a salt bridge to the target DNAbb (DNA phosphate backbone) towards base pairs 22-23 that may be affected. FIG. 14E shows one view of a combination of the deletion of P at 793 and the A708K substitution. FIG. 14F shows an alternate view, that shows that the effects of individual mutants are additive and single mutants can be combined (stacked) for even greater improvements. Arrows indicate the locations of mutations throughout FIG. 14A-14F.

FIG. 15 is a plot showing the identification of optimal Planctomycetes CasX PAM and spacers for genes of interest, as described in Example 6. On the Y-axis, percent GFP negative cells, indicating cleavage of a GFP reporter, is shown. On the X-axis, different PAM sequences and spacers: ATC PAM, CTC PAM and TTC PAM. GTC, TTT and CTT PAMs were also tested and showed no activity.

FIG. 16 is a plot showing that improved CasX variants generated by DME can edit both canonical and non-canonical PAMs more efficiently than reference CasX proteins, as described in Example 6. The Y-axis shows the average fold improvement in editing relative to a reference sgRNA:protein pair (SEQ ID NO:2, SEQ ID NO: 5) with 2 targets, N=6. Protein variants, from left to right for each set of bars were: A708K+[P793]+A739V; L379R+A708K+[P793]; C477K+A708K+[P793]; L379R+C477K+A708K+[P793]; L379R+A708K+[P793]+A739V; C477K+A708K+[P793]+A739V; and L379R+C477K+A708K+[P793]+A739V. Reference CasX and protein variants were assayed with a reference sgRNA scaffold of SEQ ID NO: 5 with DNA encoding spacer sequences of, from left to right, E6 (SEQ ID NO: 29) with a TTC PAM; E7 (SEQ ID NO: 30) with a TTC PAM; GFP8 (SEQ ID NO: 31) with a TTC PAM; B1 (SEQ ID NO: 32) with a CTC PAM and A7 (SEQ ID NO: 33) with an ATC PAM.

FIGS. 17A-17F are a series of plots showing that a reference CasX protein and a reference sgRNA scaffold pair is highly specific for the target sequence, as described in Example 7. FIG. 17A and FIG. 17D, Streptococcus pyogenes Cas9 (SpyCas9) was assayed with two different gNA spacers and a 5′ PAM site (SEQ ID NOs: 34-65) and (SEQ ID NOs: 136-166) for its ability to edit templates with a target sequence complementary to the spacer sequence (arrow), or with 1, 2, 3 or 4 mutations in the target sequence relative to the spacer sequence. FIG. 17B and FIG. 17E, Staphylococcus aureus Cas9 (SauCas9) was assayed with two different gNA spacers and a 5′ PAM site (SEQ ID NOs: 66-103) and (SEQ ID NOs: 167-204) for its ability to edit templates with a target sequence complementary to the spacer sequence (arrow), or with 1, 2, 3 or 4 mutations in the target sequence relative to the spacer sequence. FIG. 17C and FIG. 17F, the reference Plm CasX protein and sgNA scaffold pair was assayed with two different gNA spacers and a 3′ PAM site (SEQ ID NOs: 104-135) and (SEQ ID NOs: 205-236) for its ability to edit templates with a target sequence complementary to the spacer sequence (arrow), or with 1, 2, 3 or 4 mutations in the target sequence relative to the spacer sequence. In all of FIG. 17A-17F, the X-axis shows the fraction of cells where gene editing at the target sequence occurred.

FIG. 18 illustrates a scaffold stem loop of an exemplary reference sgRNA of the disclosure (SEQ ID NO: 237).

FIG. 19 illustrates an extended stem loop sequence of an exemplary reference sgRNA of the disclosure (SEQ ID NO: 238).

FIGS. 20A-20B are a pair of plots that demonstrate that specific subsets of changes discovered by DME of the CasX are more likely to predict improvements of activity, as described in Example 4. The plots represent data from the experiments described in FIG. 7 and FIG. 8. FIG. 20A shows that changing amino acids within a distance of 10 Angstroms (A) of the guide RNA to hydrophobic residues (A, V, I, L, M, F, Y, W) results in a significantly less active protein. FIG. 20B demonstrates that, in contrast, changing a residue within 10 A of the RNA to a positively charged amino acid (R, H, K) is likely to improve activity.

FIG. 21 illustrates an alignment of two reference CasX protein sequences (SEQ ID NO: 1, top: SEQ ID NO: 2, bottom), with domains annotated.

FIG. 22 illustrates the domain organization of a reference CasX protein of SEQ ID NO: 1. The domains have the following coordinates: non-target strand binding (NTSB) domain: amino acids 101-191; Helical I domain: amino acids 57-100 and 192-332; Helical II domain: 333-509; oligonucleotide binding domain (OBD): amino acids 1-56 and 510-660; RuvC DNA cleavage domain (RuvC): amino acids 551-824 and 935-986, target strand loading (TSL) domain: amino acids 825-934. Note that the Helical I, OBD and RuvC domains are non-contiguous.

FIG. 23 illustrates an alignment of two CasX reference sgRNA scaffolds SEQ ID NO: 5 (top) and SEQ ID NO: 4 (bottom).

FIG. 24 shows an SDS-PAGE gel of StX2 (CasX reference of SEQ ID NO: 2) purification fractions visualized by colloidal Coomassie staining, as described in Example 8. The lanes, from left to right, are: Pellet: insoluble portion following cell lysis, Lysate: soluble portion following cell lysis, Flow Thru: protein that did not bind the heparin column, Wash: protein that eluted from the column in wash buffer, Elution: protein eluted from the heparin column with elution buffer, Flow Thru: Protein that did not bind the StrepTactin column, Elution: protein eluted from the StrepTactin column with elution buffer, Injection: concentrated protein injected onto the s200 gel filtration column, Frozen: pooled fractions from the s200 elution that have been concentrated and frozen.

FIG. 25 shows the chromatogram from a size exclusion chromatography assay of the StX2, as described in Example 8.

FIG. 26 shows an SDS-PAGE gel of StX2 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. From right to left: Injection sample, molecular weight markers, lanes 3-9: samples from the indicated elution volumes.

FIG. 27 shows the chromatogram from a size exclusion chromatography assay of the CasX 119, using of Superdex 200 16/600 pg gel filtration, as described in Example 8. The 67.47 mL peak corresponds to the apparent molecular weight of CasX variant 119 and contained the majority of CasX variant 119 protein.

FIG. 28 shows an SDS-PAGE gel of CasX 119 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. Samples from the indicated fractions were resolved by SDS-PAGE and stained with colloidal Coomassie. From right to left, Injection: sample of protein injected onto the gel filtration column, molecular weight markers, lanes 3-10: samples from the indicated elution volumes.

FIG. 29 shows an SDS-PAGE gel of purification samples of CasX 438, visualized on a Bio-Rad Stain-Free™ gel. The lanes, from left to right, are: Pellet: insoluble portion following cell lysis, Lysate: soluble portion following cell lysis. Flow Thru: protein that did not bind the heparin column, Elution: protein eluted from the heparin column with elution buffer, Flow Thru: Protein that did not bind the StrepTactin column, Elution: protein eluted from the StrepTactin column with elution buffer, Injection: concentrated protein injected onto the s200 gel filtration column, Pool: pooled CasX-containing fractions, Final: pooled fractions from the s200 elution that have been concentrated and frozen.

FIG. 30 shows the chromatogram from a size exclusion chromatography assay of the CasX 438, using of Superdex 200 16/600 pg gel filtration, as described in Example 8. The 69.13 mL peak corresponds to the apparent molecular weight of CasX variant 438 and contained the majority of CasX variant 438 protein.

FIG. 31 shows an SDS-PAGE gel of CasX 438 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. Samples from the indicated fractions were resolved by SDS-PAGE and stained with colloidal Coomassie. From right to left, Injection: sample of protein injected onto the gel filtration column, molecular weight markers, lanes 3-10: samples from the indicated elution volumes.

FIG. 32 shows an SDS-PAGE gel of purification samples of CasX 457, visualized on a Bio-Rad Stain-Free™ gel. The lanes, from left to right, are: Pellet: insoluble portion following cell lysis, Lysate: soluble portion following cell lysis, Flow Thru: protein that did not bind the heparin column, Wash, Elution: protein eluted from the heparin column with elution buffer, Flow Thru: Protein that did not bind the StrepTactin column, Elution: protein eluted from the StrepTactin column with elution buffer, Injection: concentrated protein injected onto the s200 gel filtration column, Final: pooled fractions from the s200 elution that have been concentrated and frozen.

FIG. 33 shows the chromatogram from a size exclusion chromatography assay of the CasX 457, using of Superdex 200 16/600 pg gel filtration, as described in Example 8 The 67.52 mL peak corresponds to the apparent molecular weight of CasX variant 457 and contained the majority of CasX variant 457 protein.

FIG. 34 shows an SDS-PAGE gel of CasX 457 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. Samples from the indicated fractions were resolved by SDS-PAGE and stained with colloidal Coomassie. From right to left, Injection: sample of protein injected onto the gel filtration column, molecular weight markers, lanes 3-10: samples from the indicated elution volumes.

FIG. 35 is a schematic showing the organization of the components in the pSTX34 plasmid used to assemble the CasX constructs, as described in Example 9.

FIG. 36 is a schematic showing the steps of generating the CasX 119 variant, as described in Example 9.

FIG. 37 is a graph of the results of an assay for the quantification of active fractions of RNP formed by sgRNA174 and the CasX variants 119 and 457, as described in Example 19. Equimolar amounts of RNP and target were co-incubated and the amount of cleaved target was determined at the indicated timepoints. Mean and standard deviation of three independent replicates are shown for each timepoint. The biphasic fit of the combined replicates is shown. “2” refers to the reference CasX protein of SEQ ID NO: 2.

FIG. 38 is a graph of the results of an assay for quantification of active fractions of RNP formed by CasX2 and reference guide 2 the modified sgRNA guides 32, 64, and 174, as described in Example 19. Equimolar amounts of RNP and target were co-incubated and the amount of cleaved target was determined at the indicated timepoints. Mean and standard deviation of three independent replicates are shown for each timepoint. The biphasic fit of the combined replicates is shown. “2” refers to reference gRNAs SEQ ID NO: 5, respectively, and the identifying number of modified sgRNAs are indicated in Table 2.

FIG. 39 is a graph of the results of an assay for quantification of cleavage rates of RNP formed by sgRNA174 and the CasX variants 119 and 457, as described in Example 19. Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. Mean and standard deviation of three independent replicates are shown for each timepoint. The monophasic fit of the combined replicates is shown.

FIG. 40 is a graph of the results of an assay for quantification of cleavage rates of RNP formed by CasX2 and the sgRNA guide variants 2, 32, 64 and 174, as described in Example 19. Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. Mean and standard deviation of three independent replicates are shown for each timepoint. The monophasic fit of the combined replicates is shown.

FIG. 41 is a graph of the results of an assay for quantification of initial velocities of RNP formed by CasX2 and the sgRNA guide variants 2, 32, 64 and 174, as described in Example 19. The first two time-points of the previous cleavage experiment were fit with a linear model to determine the initial cleavage velocity.

FIG. 42 is a schematic showing an example of CasX protein and scaffold DNA sequence for packaging in adeno-associated virus (AAV), as described in Example 20. The DNA segment between the AAV inverted terminal repeats (ITRs), comprised of a CasX-encoding DNA and its promoter, and scaffold-encoding DNA and its promoter gets packaged within an AAV capsid during AAV production.

FIG. 43 is a graph showing representative results of AAV titering by qPCR, as described in Example 20. During AAV purification, flow through (FT) and consecutive eluent fractions (1-6) are collected and titered by qPCR. Most virus, ˜1e14 viral genomes in this example, is found in the second elution fraction.

FIG. 44 shows the results of an AAV-mediated gene editing experiment in the SOD1-GFP reporter cell line, as described in Example 21. CasX constructs (CasX 119 and guide 64 with SOD1 targeting spacer 2, ATGTTCATGAGTTTGGAGAT; SEQ ID NO: 239) and SauCas9 with SOD1 targeting spacer were packaged in AAV vectors and used to transduce SOD1-GFP reporter cells at a range of different multiplicity of infection (MOIs, no. of viral genomes/cell). Twelve days later, cells were assayed for GFP disruption via FACS. In this example, CasX and SauCas9 shows equivalent levels of editing, where 1-2% of the cells show GFP disruption at the highest MOIs, 1e7 or 1e6.

FIG. 45 shows the results of a second AAV-mediated gene editing experiment in the SOD1-GFP reporter cell line, as described in Example 21. CasX constructs 119.64 with SOD1 targeting spacer (2, ATGTTCATGAGTTTGGAGAT; SEQ ID NO: 239) and SauCas9 with SOD1 targeting spacer were packaged in AAV vectors and used to transduce SOD1-GFP reporter cells at a range of different multiplicity of infection (MOIs, no. of viral genomes/cell). Twelve days later, cells were assayed for GFP disruption via FACS. In this example, CasX and SauCas9 shows equivalent levels of editing at the highest MOI, where ˜2-4% of the cells show GFP disruption.

FIG. 46 shows the results of an AAV-mediated gene editing experiment in neural progenitor cells (NPCs) from the G93A mouse model of ALS, as described in Example 21. CasX constructs (CasX 119 and guide 64 with SOD1 targeting spacer 2, ATGTTCATGAGTTTGGAGAT; SEQ ID NO: 239) was packaged in an AAV vector and used to transduce G93A NPCs at a range of different multiplicity of infection (MOIs, no. of viral genomes/cell). Twelve days later, cells were assayed for gene editing via T7E1 assay. Agarose gel image from the T7E1 assay shown here demonstrates successful editing of the SOD1 locus. Double arrows show the two DNA bands as a result of successful editing in cells.

FIG. 47 shows the results of an editing assay of 6 target genes in HEK293T cells, as described in Example 23. Each dot represents results using an individual spacer.

FIG. 48 shows the results of an editing assay of 6 target genes in HEK293T cells, with individual bars representing the results obtained with individual spacers, as described in Example 23.

FIG. 49 shows the results of an editing assay of 4 target genes in HEK293T cells, as described in Example 23. Each dot represents results using an individual spacer utilizing a CTC (CTCN) PAM.

FIG. 50 is a schematic showing the steps of Deep Mutational Evolution used to create libraries of genes encoding CasX variants, as described in Example 24. The pSTX1 backbone is minimal, composed of only a high-copy number origin and KanR resistance gene, making it compatible with the recombineering E. coli strain EcNR2. pSTX2 is a BsmbI destination plasmid for aTc-inducible expression in E. coli.

FIG. 51 is dot plot graphs showing the results of CRISPRi screens for mutations in libraries D1, D2, and D3, as described in Example 24. In the absence of CRISPRi, E. coli constitutively express both GFP and RFP, resulting in intense fluorescence in both wavelengths, represented by dots in the upper-right region of the plot. CasX proteins resulting in CRISPRi of GFP can reduce green fluorescence by >10-fold, while leaving red fluorescence unaltered, and these cells fall within the indicated Sort Gate 1. The total fraction of cells exhibiting CRISPRi is indicated.

FIG. 52 is photographs of colonies grown in the ccdB assay, as described in Example 24. 10-fold dilutions were assayed in the presence of glucose or arabinose to induce expression of the ccdB toxin, resulting in approximately a 1000-fold difference between functional and nonfunctional proteins. When grown in liquid culture, the resolving power was approximately 10,000-fold, as seen on the right-hand side.

FIG. 53 is a graph of HEK iGFP genome editing efficiency testing CasX variants with sgRNA 2 (SEQ ID NO:5), with appropriate spacers, with data expressed as fold-improvement over the wild-type CasX protein (SEQ ID NO: 2) in the HEK iGFP editing assay, as described in Example 24. Single mutations are shown at the top, with groups of mutations shown at the bottom of the graph). Error bars combine internal measurement error (SD) and inter-experimental measurement error (SD across replicate experiments for those variants tested more than once), in at least triplicate assays.

FIG. 54 is a scatterplot showing results of the SOD1-GFP reporter assay for CasX variants with sgRNA scaffold 2 utilizing two different spacers for GFP, as described in Example 24.

FIG. 55 is a graph showing the results of the HEK293 iGFP genome editing assay assessing editing across four different PAM sequences comparing wild-type CasX (SEQ ID NO: 2) and CasX variant 119; both utilizing sgRNA scaffold 1 (SEQ ID NO: 4), with spacers utilizing four different PAM sequences, as described in Example 24.

FIG. 56 is a graph showing the results of genome editing activity of CasX variant 119 and sgRNA 174 compared to wild-type CasX 2 and guide scaffold 1 in the iGFP lipofection assay utilizing two different spacers, as described in Example 24.

FIG. 57 is a graph showing the results of genome editing activity of CasX variant 119 and sgRNA 174 compared to wild-type CasX and guide in the iGFP lentiviral transduction assay, using two different spacers, as described in Example 24.

FIG. 58 is a graph showing the results of genome editing in the more stringent lentiviral assay to compare the editing activity of four CasX variants (119, 438, 488 and 491) and the optimized sgNA 174 and two different spacers, as described in Example 24. The results show the step-wise improvement in editing efficiency achieved by the additional modifications and domain swaps introduced to the starting-point 119 variant.

FIGS. 59A-59B show the results of NGS analyses of the libraries of sgRNA, as described in Example 25. FIG. 59A shows the distribution of substitutions, deletions and insertions. FIG. 59B is a scatterplot showing the high reproducibility of variant representation in two separate library pools after the CRISPRi assay in the unsorted, naive population of cells. (Library pool D3 vs D2 are two different versions of the dCasX protein, and represent replicates of the CRISPRi assay.)

FIGS. 60A-60B show the structure of wild-type CasX and RNA guide (SEQ ID NO:4). FIG. 60A depicts the CryoEM structure of Deltaproteobacteria CasX protein:sgRNA RNP complex (PDB id: 6YN2), including two stem loops, a pseudoknot, and a triplex. FIG. 60B depicts the secondary structure of the sgRNA was identified from the structure shown in (A) using the tool RNAPDBee 2.0 (mapdbee.cs.put.poznan.pl/, using the tools 3DNA/DSSR, and using the VARNA visualization tool). RNA regions are indicated. Residues that were not evident in the PDB crystal structure file are indicated by plain-text letters (i.e., not encircled), and are not included in residue numbering.

FIGS. 61A-61C depict comparisons between two guide RNA scaffolds. FIG. 61A provides the sequence alignment between the single guide scaffold 1 (SEQ ID NO: 4) and scaffold 2 (SEQ ID NO: 5). FIG. 61B shows the predicted secondary structure of scaffold 1 (without the 5 ACAUCU bases which were not in the cryoEM structure). Prediction was done using RNAfold (v 2.1.7), using a constraint that was derived from the base-pairing observed in the cryoEM structure (see FIGS. 60A-60B). This constraint required the base pairs observed in the cryoEM structure to be formed, and required the bases involved in triplex formation to be unpaired. This structure has distinct base pairing from the lowest-energy predicted structure at the 5′ end (i.e., the pseudoknot and triplex loop). FIG. 61C shows the predicted secondary structure of scaffold 2. Prediction was done for scaffold 1, using a similar constraint based on the sequence alignment.

FIG. 62 shows a graph comparing GFP-knockdown capability of scaffold 1 versus scaffold 2 in GFP-lipofection assay, using four different spacers utilizing different PAM sequences, as described in Example 25. The results demonstrate the greater editing imparted by use of the modified scaffold 2 compared to the wild-type scaffold 1; the latter showing no editing with spacers utilizing GTC and CTC PAM sequences.

FIGS. 63A-63C shows graphs depicting the enrichment of single variants across the scaffold, revealing mutable regions, as described in Example 25. FIG. 63A depicts substituted bases (A, T, G, or C; top to bottom), FIG. 63B depicts inserted bases (A, T, G, or C; top to bottom), and FIG. 63C depicts deletions at the individual nucleotide position (X-axis) across scaffold 2. Enrichment values were averaged across the three dead CasX versions, relative to the average WT value. Scaffolds with relative log 2 enrichment>0 are considered ‘enriched’, as they were more represented in the sorted population relative to the naive population than the wildtype scaffold was represented. Error bars represent the confidence interval across the three catalytically dead CasX experiments.

FIG. 64 are scatterplots showing that the enrichment values obtained across different dCasX variants are largely consistent, as described in Example 25. Libraries D2 and DDD have highly correlated enrichment scores, while D3 is more distinct.

FIG. 65 shows a bar graph of cleavage activity of several scaffold variants in a more stringent lipofection assay at the SOD1-GFP locus, as described in Example 25.

FIG. 66 shows a bar graph of cleavage activity for several scaffold variants using two different spacers; 8.2 and 8.4 that target SOD1-GFP locus (and a non-targeting spacer NT), with low-MOI lentiviral transduction using a p34 plasmid backbone, as described in Example 25.

FIG. 67 is a schematic showing the secondary structure of single guide 174 on top and the linear structure on the bottom, with lines joining those segments associating by base-pairing or other non-covalent interactions. The scaffold stem (white, no fill) (and loop) and the extended stem (grey, no fill) (and loop) are adjacent from 5′ to 3′ in the sequence. However, the pseudoknot and extended stems are formed from strands that have intervening regions in the sequence. The triplex is formed, in the case of single guide 174, comprising nucleotides 5′-CUUUG′-3′ AND 5′-CAAAG-3′ that form a base-paired duplex and nucleotides 5′-UUU-3′ that associates with the 5′-AAA-3′ to form the triplex region.

FIGS. 68A and 68 B show comparisons between the highly-evolved single guide 174 and the scaffolds 1 and 2 that served as the starting points for the DME procedures described in Example 25. FIG. 68A shows a bar graph of cleavage activity of head-to-head comparisons of cleavage activity of the guide scaffolds with five different spacers in a plasmid lipofection assay at the GFP locus in HEK-GFP cells. FIG. 68B shows the sequence alignment between scaffold 2 and guide 174 (SEQ ID NO: 2238). Asterisks indicate point mutations, and the dotted box shows the entire extended stem swap.

FIGS. 69A-69B shows scatterplots of HEK-iGFP cleavage assay for scaffolds sequences relative to WT scaffold with 2 spacers; 4.76 (FIG. 69A) and 4.77 (FIG. 69B), as described in Example 25.

FIG. 70 shows a scatterplot comparing the normalized cleavage activity of several scaffolds relative to WT with 2 spacers (4.76 and 4.77), as described in Example 25. Error bars combine internal measurement error (SD) and inter-experimental measurement error (SD across replicate experiments for those variants tested more than once), in quadrature.

FIG. 71 shows a scatterplot comparing the normalized cleavage activity of multiple scaffolds relative to WT in the HEK-iGFP cleavage assay to the enrichments obtained from the CRISPRi comprehensive screen, as described in Example 25. Generally, scaffold mutations with high enrichment (>1.5) have cleavage activity comparable to or greater than WT. Two variants have high cleavage activity with low enrichment scores (C18G and TI7G); interestingly, these substitutions are at the same position as several highly enriched insertions (FIGS. 63A-63C). Labels indicate the mutations for a subset of the comparisons.

FIG. 72 shows the results of flow cytometry analysis of Cas-mediated editing at the RHO locus in APRE19 RHO-GFP cells 14 days post-transfection for the CasX variant constructs 438, 499 and 491, as described in Example 26. The points are the results of individual samples and the light dashed lines are upper and lower quartiles.

FIG. 73 shows the quantification of cleavage rates of RNP formed by sgRNA174 and the CasX variants on targets with different PAMs. Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. The monophasic fit of the combined replicates is shown.

DETAILED DESCRIPTION

While exemplary embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the inventions claimed herein. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments of the disclosure. It is intended that the claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

“Hybridizable” or “complementary” are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize to the target nucleic acid. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, ‘bubble’ and the like).

A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (e.g., a protein, RNA), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene may include regulatory sequences including, but not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. Coding sequences encode a gene product upon transcription or transcription and translation; the coding sequences of the disclosure may comprise fragments and need not contain a full-length open reading frame. A gene can include both the strand that is transcribed, e.g. the strand containing the coding sequence, as well as the complementary strand.

The term “downstream” refers to a nucleotide sequence that is located 3′ to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′ to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5′ side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

The term “regulatory element” is used interchangeably herein with the term “regulatory sequence.” and is intended to include promoters, enhancers, and other expression regulatory elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Exemplary regulatory elements include a transcription promoter such as, but not limited to, CMV, CMV+intron A, SV40, RSV, HIV-Ltr, elongation factor 1 alpha (EF1α), MMLV-ltr, internal ribosome entry site (IRES) or P2A peptide to permit translation of multiple genes from a single transcript, metallothionein, a transcription enhancer element, a transcription termination signal, polyadenylation sequences, sequences for optimization of initiation of translation, and translation termination sequences. It will be understood that the choice of the appropriate regulatory element will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

The term “promoter” refers to a DNA sequence that contains an RNA polymerase binding site, transcription start site, TATA box, and/or B recognition element and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A promoter can be proximal or distal to the gene to be transcribed. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. A promoter of the present disclosure can include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc.

The term “enhancer” refers to regulatory element DNA sequences that, when bound by specific proteins called transcription factors, regulate the expression of an associated gene. Enhancers may be located in the intron of the gene, or 5′ or 3′ of the coding sequence of the gene. Enhancers may be proximal to the gene (i.e., within a few tens or hundreds of base pairs (bp) of the promoter), or may be located distal to the gene (i.e., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisaged as within the scope of the instant disclosure.

“Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5′ or 3′ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “enhancers” and “promoters”, above).

The term “recombinant polynucleotide” or “recombinant nucleic acid” refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such can be done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids. e.g., by genetic engineering techniques.

Similarly, the term “recombinant polypeptide” or “recombinant protein” refers to a polypeptide or protein which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a protein that comprises a heterologous amino acid sequence is recombinant.

As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection; e.g., can hybridize if the sequences share sequence similarity.

“Dissociation constant”, or “K_(d)”, are used interchangeably and mean the affinity between a ligand “L” and a protein “P”; i.e., how tightly a ligand binds to a particular protein. It can be calculated using the formula K_(d)=[L][P]/[LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively.

The disclosure provides compositions and methods useful for editing a target nucleic acid sequence. As used herein “editing” is used interchangeably with “modifying” and includes but is not limited to cleaving, nicking, deleting, knocking in, knocking out, and the like.

As used herein. “homology-directed repair” (HDR) refers to the form of DNA repair that takes place during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, and uses a donor template to repair or knock-out a target DNA, and leads to the transfer of genetic information from the donor (e.g., such as the donor template) to the target. Homology-directed repair can result in an alteration of the sequence of the target nucleic acid sequence by insertion, deletion, or mutation if the donor template differs from the target DNA sequence and part or all of the sequence of the donor template is incorporated into the target DNA at the correct genomic locus.

As used herein, “non-homologous end joining” (NHEJ) refers to the repair of double-strand breaks in DNA by direct ligation of the break ends to one another without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). NHEJ often results in indels; the loss (deletion) or insertion of nucleotide sequence near the site of the double-strand break.

As used herein “micro-homology mediated end joining” (MMEJ) refers to a mutagenic DSB repair mechanism, which always associates with deletions flanking the break sites without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). MMEJ often results in the loss (deletion) of nucleotide sequence near the site of the double-strand break.

A polynucleotide or polypeptide (or protein) has a certain percent “sequence similarity” or “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity (sometimes referred to as percent similarity, percent identity, or homology) can be determined in a number of different manners. To determine sequence similarity, sequences can be aligned using the methods and computer programs that are known in the art, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410, Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

The terms “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence.

A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an “insert”, may be attached so as to bring about the replication or expression of the attached segment in a cell.

The term “naturally-occurring” or “unmodified” or “wild-type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

As used herein, a “mutation” refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wild-type or reference amino acid sequence or to a wild-type or reference nucleotide sequence.

As used herein the term “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

A “host cell,” as used herein, denotes a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which cells are used as recipients for a nucleic acid (e.g., an expression vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector.

The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

As used herein. “treatment” or “treating,” are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can also be achieved with the eradication or amelioration of one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a composition, vector, cells, etc., that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. Such effect can be transient.

As used herein, “administering” is meant as a method of giving a dosage of a composition of the disclosure to a subject.

As used herein, a “subject” is a mammal. Mammals include, but are not limited to, domesticated animals, primates, non-human primates, humans, dogs, porcine (pigs), rabbits, mice, rats and other rodents.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

I. General Methods

The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999): Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

Where a range of values is provided, it is understood that endpoints are included and that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms “a.” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

It will be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. In other cases, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is intended that all combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

II. CasX:gNA Systems

In a first aspect, the present disclosure provides CasX:gNA systems comprising a CasX protein and one or more guide nucleic acids (gNA) for use in modifying or editing a target nucleic acid, inclusive of coding and non-coding regions. The terms CasX protein and CasX are used interchangeably herein; the terms CasX variant protein and CasX variant are used interchangeably herein. The CasX protein and gNA of the CasX:gNA systems provided herein each independently may be a reference CasX protein, a CasX variant protein, a reference gNA, a gNA variant, or any combination of a reference CasX protein, reference gNA, CasX variant protein, or gNA variant. A gNA and a CasX protein, a gNA variant and CasX variant, or any combination thereof can form a complex and bind via non-covalent interactions, referred to herein as a ribonucleoprotein (RNP) complex. In some embodiments, the use of a pre-complexed CasX:gNA confers advantages in the delivery of the system components to a cell or target nucleic acid for editing of the target nucleic acid. In the RNP, the gNA can provide target specificity to the RNP complex by including a spacer sequence (targeting sequence) having a nucleotide sequence that is complementary to a sequence of a target nucleic acid. In the RNP, the CasX protein of the pre-complexed CasX:gNA provides the site-specific activity and is guided to a target site (and further stabilized at a target site) within a target nucleic acid sequence to be modified by virtue of its association with the gNA. The CasX protein of the RNP complex provides the site-specific activities of the complex such as binding, cleavage, or nicking of the target sequence by the CasX protein. Provided herein are compositions and cells comprising the reference CasX proteins, CasX variant proteins, reference gNAs, gNA variants, and CasX:gNA gene editing pairs of any combination of CasX and gNA, as well as delivery modalities comprising the CasX:gNA. In other embodiments, the disclosure provides vectors encoding or comprising the CasX:gNA pair and, optionally, donor templates for the production and/or delivery of the CasX:gNA systems. Also provided herein are methods of making CasX proteins and gNA, as well as methods of using the CasX and gNA, including methods of gene editing and methods of treatment. The CasX proteins and gNA components of the CasX:gNA and their features, as well as the delivery modalities and the methods of using the compositions are described more fully, below.

The donor templates of the CasX:gNA systems are designed depending on whether they are utilized to correct mutations in a target gene or insert a transgene at a different locus in the genome (a “knock-in”), or are utilized to disrupt the expression of a gene product that is aberrant; e.g., it comprises one or more mutations reducing expression of the gene product or rendering the protein dysfunctional (a “knock-down” or “knock-out”). In some embodiments, the donor template is a single stranded DNA template or a single stranded RNA template. In other embodiments, the donor template is a double stranded DNA template. In some embodiments, the CasX:gNA systems utilized in the editing of the target nucleic acid comprises a donor template having all or at least a portion of an open reading frame of a gene in the target nucleic acid for insertion of a corrective, wild-type sequence to correct a defective protein. In other cases, the donor template comprises all or a portion of a wild-type gene for insertion at a different locus in the genome for expression of the gene product. In still other cases, a portion of the gene can be inserted upstream (′5) of the mutation in the target nucleic acid, wherein the donor template gene portion spans to the C-terminus of the gene, resulting, upon its insertion into the target nucleic acid, in expression of the gene product. In other embodiments, the donor template can comprise one or more mutations in an encoding sequence compared to a normal, wild-type sequence of the target gene utilized for insertion for either knocking out or knocking down (described more fully, below) the defective target nucleic acid sequence. In other embodiments, the donor template can comprise regulatory elements, an intron, or an intron-exon junction having sequences specifically designed to knock-down or knock-out a defective gene or, in the alternative, to knock-in a corrective sequence to permit the expression of a functional gene product. In some embodiments, the donor polynucleotide comprises at least about 10, at least about 20, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 10,000, at least about 15,000, at least about 25,000, at least about 50,000, at least about 100,000 or at least about 200.000 nucleotides. Provided that there are stretches of DNA sequence with sufficient numbers of nucleotides having sufficient homology flanking the cleavage site(s) of the target nucleic acid sequence targeted by the CasX:gNA (i.e., 5′ and 3′ to the cleavage site) to support homology-directed repair (the flanking regions being “homologous arms”), use of such donor templates can result in its integration into the target nucleic acid by HDR. In other cases, the donor template can be inserted by non-homologous end joining (NHEJ; which does not require homologous arms) or by microhomology-mediated end joining (MMEJ; which requires short regions of homology on the 5′ and 3′ ends). In some embodiments, the donor template comprises homologous arms on the 5′ and 3′ ends, each having at least about 2, at least about 10, at least about 20, at least about 30, at least about 50, at least about 100, at least about 150, at least about 300, at least about 1000, at least about 1500 or more nucleotides having homology with the sequences flanking the intended cleave site(s) of the target nucleic acid. In some embodiments, the CasX:gNA systems utilize two or more gNA with targeting sequences complementary to overlapping or different regions of the target nucleic acid such that the defective sequence can be excised by multiple double-stranded breaks or by nicking in locations flanking the defective sequence and the donor template inserted by HDR to replace the excised sequence. In the foregoing, the gNA would be designed to contain targeting sequences that are 5′ and 3′ to the individual site or sequence to be excised. By such appropriate selection of the targeting sequences of the gNA, defined regions of the target nucleic acid can be edited using the CasX:gNA systems described herein.

III. Guide Nucleic Acids of the CasX:gNA Systems

In other aspects, the disclosure provides guide nucleic acids (gNA) utilized in the CasX:gNA systems, and have utility in editing of a target nucleic acid. The present disclosure provides specifically-designed gNAs with targeting sequences (or “spacers”) that are complementary to (and are therefore able to hybridize with) the target nucleic acid as a component of the gene editing CasX:gNA systems. It is envisioned that in some embodiments, multiple gNAs (e.g., multiple gRNAs) are delivered by the CasX:gNA system for the modification of different regions of a gene, including regulatory elements, an exon, an intron, or an intron-exon junction. In some embodiments, the targeting sequence of the gNA is complementary to a sequence comprising one or more single nucleotide polymorphisms (SNPs) of the target nucleic. In other embodiments, the targeting sequence of the gNA is complementary to a sequence of an intergenic region. For example, when a deletion of a protein-encoding gene is desired, a pair of gNAs with targeting sequences to different or overlapping regions of the target nucleic acid sequence can be used in order to bind and cleave at two different sites within the gene that can then be edited by indel formation or homology-directed repair (HDR), which, in the case of HDR, utilizes a donor template that is inserted to replace the deleted sequence to complete the editing.

a. Reference gNA and gNA Variants

In some embodiments, a gNA of the present disclosure comprises a sequence of a naturally-occurring gNA (“reference gNA”). In other cases, a reference gNA of the disclosure may be subjected to one or more mutagenesis methods, such as the mutagenesis methods described herein, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping, in order to generate one or more gNA variants with enhanced or varied properties relative to the reference gNA. gNA variants also include variants comprising one or more exogenous sequences, for example fused to either the 5′ or 3′ end, or inserted internally. The activity of reference gNAs may be used as a benchmark against which the activity of gNA variants are compared, thereby measuring improvements in function or other characteristics of the gNA variants. In other embodiments, a reference gNA may be subjected to one or more deliberate, targeted mutations in order to produce a gNA variant, for example a rationally-designed variant. As used herein, the terms gNA, gRNA, and gDNA cover naturally-occurring molecules (reference molecules), as well as sequence variants.

In some embodiments, the gNA is a deoxyribonucleic acid molecule (“gDNA”); in some embodiments, the gNA is a ribonucleic acid molecule (“gRNA”), and in other embodiments, the gNA is a chimera, and comprises both DNA and RNA.

The gNAs of the disclosure comprise two segments; a targeting sequence and a protein-binding segment (which constitutes the scaffold, discussed herein). The targeting segment of a gNA includes a nucleotide sequence (referred to interchangeably herein as a guide sequence, a spacer, a targeting sequence, or a targeting region) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.), described more fully below.

The targeting sequence of a gNA is capable of binding to a target nucleic acid sequence, including a coding sequence, a complement of a coding sequence, a non-coding sequence, and to regulatory elements. The protein-binding segment (or “protein-binding sequence”) interacts with (e.g., binds to) a CasX protein. The protein-binding segment is alternatively referred to herein as a “scaffold”. In some embodiments, the targeting sequence and scaffold each include complementary stretches of nucleotides that hybridize to one another to form a double stranded duplex (e.g. dsRNA duplex for a gRNA). Site-specific binding and/or cleavage of a target nucleic acid sequence (e.g., genomic DNA) by the CasX:gNA can occur at one or more locations of a target nucleic acid, determined by base-pairing complementarity between the targeting sequence of the gNA and the target nucleic acid sequence.

The gNA provides target specificity to the complex by having a nucleotide sequence that is complementary to a target sequence of a target nucleic acid. The CasX of the complex provides the site-specific activities of the complex such as binding, cleavage, or nicking of the target sequence of the target nucleic acid by the CasX nuclease and/or an activity provided by a fusion partner in case of a CasX containing fusion protein, described below. In some embodiments, the disclosure provides gene editing pairs of a CasX and gNA of any of the embodiments described herein that are capable of being bound together prior to their use for gene editing and, thus, are “pre-complexed” as the RNP. The use of a pre-complexed RNP confers advantages in the delivery of the system components to a cell or target nucleic acid sequence for editing of the target nucleic acid sequence. The CasX protein of the RNP provides the site-specific activity that is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence by virtue of its association with the guide RNA comprising a targeting sequence.

In some embodiments, wherein the gNA is a gRNA, the term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: “CRISPR RNA”) of a CasX dual guide RNA (dgRNA). In a single guide RNA (sgRNA), the “activator” and the “targeter” are linked together, e.g., by intervening nucleotides). Thus, for example, a guide RNA (dgRNA or sgRNA) comprises a guide sequence and a duplex-forming segment of a crRNA, which can also be referred to as a crRNA repeat. Because the targeter sequence of a guide sequence hybridizes with a specific target nucleic acid sequence, a targeter can be modified by a user to hybridize with a desired target nucleic acid sequence. In some embodiments, the sequence of a targeter may often be a non-naturally occurring sequence. The targeter and the activator each have a duplex-forming segment, where the duplex forming segment of the targeter and the duplex-forming segment of the activator have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). In some embodiments, a targeter comprises both the guide sequence of the CasX guide RNA and a stretch of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the gNA. A corresponding tracrRNA-like molecule (the activator “trans-acting CRISPR RNA”) also comprises a duplex-forming stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the CasX guide RNA. In some cases the activator comprises one or more stem loops that can interact with CasX protein. Thus, a targeter and an activator, as a corresponding pair, hybridize to form a CasX dual guide NA, referred to herein as a “dual guide NA”, a “dgNA”, a “double-molecule guide NA”, or a “two-molecule guide NA”.

In some embodiments, the activator and targeter of the reference gNA are covalently linked to one another and comprise a single molecule, referred to herein as a “single-molecule guide NA,” “one-molecule guide NA,” “single guide NA”, “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, a “single guide DNA”, a “single-molecule DNA,” or a “one-molecule guide DNA”. (“sgNA”, “sgRNA”, or a “sgDNA”). In some embodiments, the sgNA includes an “activator” or a “targeter” and thus can be an “activator-RNA” and a “targeter-RNA,” respectively.

The reference gRNAs of the disclosure comprise four distinct regions, or domains: the RNA triplex, the scaffold stem, the extended stem, and the targeting sequence (specific for a target nucleic acid. The RNA triplex, the scaffold stem, and the extended stem, together, are referred to as the “scaffold” of the reference gNA, based upon which further gNA variants are generated.

b. RNA Triplex

In some embodiments of the guide NAs provided herein, the gNA comprises an RNA triplex, and the RNA triplex comprises the sequence of a UUU--Nx(˜4-15)--UUU stem loop (SEQ ID NO: 241) that ends with an AAAG after 2 intervening stem loops (the scaffold stem loop and the extended stem loop), forming a pseudoknot that may also extend past the triplex into a duplex pseudoknot. The UU-UUU-AAA sequence of the triplex forms as a nexus between the targeting sequence, scaffold stem, and extended stem. In exemplary gRNAs, the UUU-loop-UUU region is coded for first, then the scaffold stem loop, and then the extended stem loop, which is linked by the tetraloop, and then an AAAG closes off the triplex before becoming the targeting sequence.

c. Scaffold Stem Loop

In some embodiments of gNAs of the disclosure, the triplex region is followed by the scaffold stem loop. The scaffold stem loop is a region of the gNA that is bound by CasX protein (such as a reference or CasX variant protein). In some embodiments, the scaffold stem loop is a fairly short and stable stem loop, and increases the overall stability of the gNA. In some cases, the scaffold stem loop does not tolerate many changes, and requires some form of an RNA bubble. In some embodiments, the scaffold stem is necessary for gNA function. While it is perhaps analogous to the nexus stem of Cas9 as being a critical stem loop, the scaffold stem of a gNA, in some embodiments, has a necessary bulge (RNA bubble) that is different from many other stem loops found in CRISPR/Cas systems. In some embodiments, the presence of this bulge is conserved across gNA that interact with different CasX proteins. An exemplary sequence of a scaffold stem loop sequence of a gNA comprises the sequence CCAGCGACUAUGUCGUAUGG (SEQ ID NO: 242). In other embodiments, the disclosure provides gNA variants wherein the scaffold stem loop is replaced with an RNA stem loop sequence from a heterologous RNA source with proximal 5′ and 3′ ends, such as, but not limited to stem loop sequences selected from MS2, Qβ, U1 hairpin II, Uvsx, or PP7 stem loops. In some cases, the heterologous RNA stem loop of the gNA is capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule.

d. Extended Stem Loop

In some embodiments of the gNAs of the disclosure, the scaffold stem loop is followed by the extended stem loop. In some embodiments, the extended stem comprises a synthetic tracr and crRNA fusion that is largely unbound by the CasX protein. In some embodiments, the extended stem loop can be highly malleable. In some embodiments, a single guide gRNA is made with a GAAA tetraloop linker or a GAGAAA linker between the tracr and crRNA in the extended stem loop. In some cases, the targeter and activator of a sgNA are linked to one another by intervening nucleotides and the linker can have a length of from 3 to 20 nucleotides. In some embodiments of the sgNAs of the disclosure, the extended stem is a large 32-bp loop that sits outside of the CasX protein in the ribonucleoprotein complex. An exemplary sequence of an extended stem loop sequence of a sgNA comprises the sequence GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC (SEQ ID NO: 15). In some embodiments, the extended stem loop comprises a GAGAAA spacing sequence. In some embodiments, the disclosure provides gNA variants wherein the extended stem loop is replaced with an RNA stem loop sequence from a heterologous RNA source with proximal 5′ and 3′ ends, such as, but not limited to stem loop sequences selected from MS2, Qβ, U1 hairpin II, Uvsx, or PP7 stem loops. In such cases, the heterologous RNA stem loop increases the stability of the gNA. In other embodiments, the disclosure provides gNA variants having an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides.

e. Targeting Sequence

In some embodiments of the gNAs of the disclosure, the extended stem loop is followed by a region that forms part of the triplex, and then the targeting sequence (or “spacer”). The targeting sequence can be designed to target the CasX ribonucleoprotein holo complex to a specific region of the target nucleic acid sequence. Thus, the gNA targeting sequences of the gNAs of the disclosure have sequences complementarity to, and therefore can hybridize to, a portion of the target nucleic acid in a nucleic acid in a eukaryotic cell, (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.) as a component of the RNP when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5′ to the non-target strand sequence complementary to the target sequence.

In some embodiments, the disclosure provides a gNA wherein the targeting sequence of the gNA is complementary to a target nucleic acid sequence comprising one or more mutations compared to a wild-type gene sequence for purposes of editing the sequence comprising the mutations with the CasX:gNA systems of the disclosure. In some embodiments, the targeting sequence of a gNA is designed to be specific for an exon of the gene of the target nucleic acid. In other embodiments, the targeting sequence of a gNA is designed to be specific for an intron of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gNA is designed to be specific for an intron-exon junction of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gNA is designed to be specific for a regulatory element of the gene of the target nucleic acid. In some embodiments, the targeting sequence of the gNA is designed to be complementary to a sequence comprising one or more single nucleotide polymorphisms (SNPs) in a gene of the target nucleic acid. SNPs that are within the coding sequence or within non-coding sequences are both within the scope of the instant disclosure. In other embodiments, the targeting sequence of the gNA is designed to be complementary to a sequence of an intergenic region of the gene of the target nucleic acid.

In some embodiments, the targeting sequence of a gNA is designed to be specific for a regulatory element that regulates expression of the gene product of the target nucleic acid. Such regulatory elements include, but are not limited to promoter regions, enhancer regions, intergenic regions, 5′ untranslated regions (5′ UTR), 3′ untranslated regions (3′ UTR), conserved elements, and regions comprising cis-regulatory elements. The promoter region is intended to encompass nucleotides within 5 kb of the initiation point of the encoding sequence or, in the case of gene enhancer elements or conserved elements, can be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the encoding sequence of the gene of the target nucleic acid. In some embodiments of the foregoing, the targets are those in which the encoding gene of the target is intended to be knocked out or knocked down such that the encoded protein comprising mutations is not expressed or is expressed at a lower level in a cell.

In some embodiments, the targeting sequence of a gNA has between 14 and 35 consecutive nucleotides. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides. In some embodiments, the targeting sequence of the gNA consists of 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides and the targeting sequence can comprise 0 to 5, 0 to 4, 0 to 3, or 0 to 2 mismatches relative to the target nucleic acid sequence and retain sufficient binding specificity such that the RNP comprising the gNA comprising the targeting sequence can form a complementary bond with respect to the target nucleic acid.

In some embodiments, the CasX:gNA system comprises a first gNA and further comprises a second (and optionally a third, fourth, fifth, or more) gNA, wherein the second gNA or additional gNA has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid sequence compared to the targeting sequence of the first gNA such that multiple points in the target nucleic acid are targeted, and for example, multiple breaks are introduced in the target nucleic acid by the CasX. It will be understood that in such cases, the second or additional gNA is complexed with an additional copy of the CasX protein. By selection of the targeting sequences of the gNA, defined regions of the target nucleic acid sequence bracketing a mutation can be modified or edited using the CasX:gNA systems described herein, including facilitating the insertion of a donor template.

f. gNA Scaffolds

With the exception of the targeting sequence region, the remaining regions of the gNA are referred to herein as the scaffold. In some embodiments, the gNA scaffolds are derived from naturally-occurring sequences, described below as reference gNA. In other embodiments, the gNA scaffolds are variants of reference gNA wherein mutations, insertions, deletions or domain substitutions are introduced to confer desirable properties on the gNA.

In some embodiments, a reference gRNA comprises a sequence isolated or derived from Deltaproteobacteria. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary CasX reference tracrRNA sequences isolated or derived from Deltaproteobacteria may include: ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU AUGGACGAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 6) and ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU AUGGACGAAGCGCUUAUUUAUCGG (SEQ ID NO: 7). Exemplary crRNA sequences isolated or derived from Deltaproteobacteria may comprise a sequence of CCGAUAAGUAAAACGCAUCAAAG (SEQ ID NO: 243). In some embodiments, a reference gNA comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical or 100% identical to a sequence isolated or derived from Deltaproteobacteria.

In some embodiments, a reference guide RNA comprises a sequence isolated or derived from Planctomycetes. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary reference tracrRNA sequences isolated or derived from Planctomycetes may include: UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA UGGGUAAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 8) and UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA UGGGUAAAGCGCUUAUUUAUCGG (SEQ ID NO: 9). Exemplary crRNA sequences isolated or derived from Planctomycetes may comprise a sequence of UCUCCGAUAAAUAAGAAGCAUCAAAG (SEQ ID NO: 244). In some embodiments, a reference gNA comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical or 100% identical to a sequence isolated or derived from Planctomycetes.

In some embodiments, a reference gNA comprises a sequence isolated or derived from Candidatus Sungbacteria. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary CasX reference tracrRNA sequences isolated or derived from Candidatus Sungbacteria may comprise sequences of: GUUUACACACUCCCUCUCAUAGGGU (SEQ ID NO: 10), GUUUACACACUCCCUCUCAUGAGGU (SEQ ID NO: 11), UUUUACAUACCCCCUCUCAUGGGAU (SEQ ID NO: 12) and GUUUACACACUCCCUCUCAUGGGGG (SEQ ID NO: 13). In some embodiments, a reference guide RNA comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical or 100% identical to a sequence isolated or derived from Candidatus Sungbacteria.

Table 1 provides the sequences of reference gRNA tracr, cr and scaffold sequences. In some embodiments, the disclosure provides gNA sequences wherein the gNA has a scaffold comprising a sequence having at least one nucleotide modification relative to a reference gNA sequence having a sequence of any one of SEQ ID NOS: 4-16 of Table 1. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gNA, or where a gNA is a gDNA or a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gNA sequence embodiments described herein.

TABLE 1 Reference gRNA tracr, cr and scaffold sequences SEQ ID NO. Nucleotide Sequence  4 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGA CUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAU AAGUAAAACGCAUCAAAG  5 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGAC UAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAU AAAUAAGAAGCAUCAAAG  6 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGA CUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA  7 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGA CUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGG  8 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGAC UAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGA  9 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGAC UAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG 10 GUUUACACACUCCCUCUCAUAGGGU 11 GUUUACACACUCCCUCUCAUGAGGU 12 UUUUACAUACCCCCUCUCAUGGGAU 13 GUUUACACACUCCCUCUCAUGGGGG 14 CCAGCGACUAUGUCGUAUGG 15 GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC 16 GGCGCUUUUAUCUCAUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAUGGGUAAAGCGCUUAUUUAUCGGA

g. gNA Variants

In another aspect, the disclosure relates to guide nucleic acid variants (referred to herein alternatively as “gNA variant” or “gRNA variant”), which comprise one or more modifications relative to a reference gRNA scaffold. As used herein, “scaffold” refers to all parts to the gNA necessary for gNA function with the exception of the spacer sequence.

In some embodiments, a gNA variant comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced regions relative to a reference gRNA sequence of the disclosure. In some embodiments, a mutation can occur in any region of a reference gRNA scaffold to produce a gNA variant. In some embodiments, the scaffold of the gNA variant sequence has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

In some embodiments, a gNA variant comprises one or more nucleotide changes within one or more regions of the reference gRNA scaffold that improve a characteristic of the reference gRNA. Exemplary regions include the RNA triplex, the pseudoknot, the scaffold stem loop, and the extended stem loop. In some cases, the variant scaffold stem further comprises a bubble. In other cases, the variant scaffold further comprises a triplex loop region. In still other cases, the variant scaffold further comprises a 5′ unstructured region. In some embodiments, the gNA variant scaffold comprises a scaffold stem loop having at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the gNA variant scaffold comprises a scaffold stem loop having at least 60% sequence identity to SEQ ID NO: 14. In other embodiments, the gNA variant comprises a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 245). In other embodiments, the disclosure provides a gNA scaffold comprising, relative to SEQ ID NO:5, a C18G substitution, a G55 insertion, a U1 deletion, and a modified extended stem loop in which the original 6 nt loop and 13 most-loop-proximal base pairs (32 nucleotides total) are replaced by a Uvsx hairpin (4 nt loop and 5 loop-proximal base pairs; 14 nucleotides total) and the loop-distal base of the extended stem was converted to a fully base-paired stem contiguous with the new Uvsx hairpin by deletion of the A99 and substitution of G65U. In the foregoing embodiment, the gNA scaffold comprises the sequence ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG GGAGCAUCAAAG (SEQ ID NO: 2238).

All gNA variants that have one or more improved characteristics, or add one or more new functions when the variant gNA is compared to a reference gRNA described herein, are envisaged as within the scope of the disclosure. A representative example of such a gNA variant is guide 174 (SEQ ID NO: 2238), the design of which is described in the Examples. In some embodiments, the gNA variant adds a new function to the RNP comprising the gNA variant. In some embodiments, the gNA variant has an improved characteristic selected from: improved stability; improved solubility; improved transcription of the gNA; improved resistance to nuclease activity; increased folding rate of the gNA; decreased side product formation during folding; increased productive folding; improved binding affinity to a CasX protein; improved binding affinity to a target DNA when complexed with a CasX protein; improved gene editing when complexed with a CasX protein; improved specificity of editing when complexed with a CasX protein; and improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA when complexed with a CasX protein, and any combination thereof. In some cases, the one or more of the improved characteristics of the gNA variant is at least about 1.1 to about 100,000-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more improved characteristics of the gNA variant is at least about 1.1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000-fold or more improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more of the improved characteristics of the gNA variant is about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100.00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more improved characteristics of the gNA variant is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

In some embodiments, a gNA variant can be created by subjecting a reference gNA to a one or more mutagenesis methods, such as the mutagenesis methods described herein, below, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping, in order to generate the gNA variants of the disclosure. The activity of reference gNAs may be used as a benchmark against which the activity of gNA variants are compared, thereby measuring improvements in function of gNA variants. In other embodiments, a reference gNA may be subjected to one or more deliberate, targeted mutations, substitutions, or domain swaps in order to produce a gNA variant, for example a rationally designed variant. Exemplary gNA variants produced by such methods are described in the Examples and representative sequences of gNA scaffolds are presented in Table 2.

In some embodiments, the gNA variant comprises one or more modifications compared to a reference guide nucleic acid scaffold sequence, wherein the one or more modification is selected from: at least one nucleotide substitution in a region of the reference gNA at least one nucleotide deletion in a region of the reference gNA; at least one nucleotide insertion in a region of the reference gNA; a substitution of all or a portion of a region of the reference gNA; a deletion of all or a portion of a region of the reference gNA; or any combination of the foregoing. In some cases, the modification is a substitution of 1 to 15 consecutive or non-consecutive nucleotides in the reference gNA in one or more regions. In other cases, the modification is a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the reference gNA in one or more regions. In other cases, the modification is an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the reference gNA in one or more regions. In other cases, the modification is a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5′ and 3′ ends. In some cases, a gNA variant of the disclosure comprises two or more modifications in one region relative to a reference gRNA. In other cases, a gNA variant of the disclosure comprises modifications in two or more regions. In other cases, a gNA variant comprises any combination of the foregoing modifications described in this paragraph. In some embodiments, exemplary modifications of gNA of the disclosure include the modifications of Table 24.

In some embodiments, a 5′ G is added to a gNA variant sequence, relative to a reference gRNA, for expression in vivo, as transcription from a U6 promoter is more efficient and more consistent with regard to the start site when the +1 nucleotide is a G. In other embodiments, two 5′ Gs are added to generate a gNA variant sequence for in vitro transcription to increase production efficiency, as T7 polymerase strongly prefers a G in the +1 position and a purine in the +2 position. In some cases, the 5′ G bases are added to the reference scaffolds of Table 1. In other cases, the 5′ G bases are added to the variant scaffolds of Table 2.

Table 2 provides exemplary gNA variant scaffold sequences of the disclosure. In Table 2, (−) indicates a deletion at the specified position(s) relative to the reference sequence of SEQ ID NO: 5, (+) indicates an insertion of the specified base(s) at the position indicated relative to SEQ ID NO: 5, (:) indicates the range of bases at the specified start:stop coordinates of a deletion or substitution relative to SEQ ID NO: 5, and multiple insertions, deletions or substitutions are separated by commas; e.g., A14C, T17G. In some embodiments, the gNA variant scaffold comprises any one of the sequences listed in Table 2, SEQ ID NOS: 2101-2280, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gNA, or where a gNA is a gDNA or a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gNA sequence embodiments described herein.

TABLE 2 Exemplary gNA Variant Scaffold Sequences SEQ ID NAME or NO: Modification NUCLEOTIDE SEQUENCE 2101 phage replication UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG stable GGUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG 2102 Kissing loop_b1 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUGCUCGACGCGUCCUCGAGCAGAAGCAUCAAAG 2103 Kissing loop_a UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUGCUCGCUCCGUUCGAGCAGAAGCAUCAAAG 2104 32, uvsX hairpin GUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG 2105 PP7 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCAGGAGUUUCUAUGGAAACCCUGAAGCAUCAAAG 2106 64, trip mut, GUACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU extended stem GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG truncation 2107 hyperstable UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG tetraloop GGUAAAGCGCUGCGCUUGCGCAGAAGCAUCAAAG 2108 C18G UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2109 T17G UACUGGCGCUUUUAUCGCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2110 CUUCGG loop UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG 2111 MS2 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCACAUGAGGAUUACCCAUGUGAAGCAUCAAAG 2112 -1, A2G, -78, GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG G77T GUAAAGCGCUUAUUUAUCGUGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2113 QB UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUGCAUGUCUAAGACAGCAGAAGCAUCAAAG 2114 45, 44 hairpin UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCAGGGCUUCGGCCGAAGCAUCAAAG 2115 U1A UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCAAUCCAUUGCACUCCGGAUUGAAGCAUCAAAG 2116 A14C, T17G UACUGGCGCUUUUCUCGCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2117 CUUCGG loop UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG modified GGUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG 2118 Kissing loop_b2 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUGCUCGUUUGCGGCUACGAGCAGAAGCAUCAAAG 2119 -76:78, -83:87 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGAGAGAUAAAUAAGAAGCAUCAAAG 2120 -4 UACGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG GUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2121 extended stem UACUGGCGCCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU truncation GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2122 C55 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUC GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2123 trip mut UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUC GGUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG 2124 -76:78 UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUC GGUAAAGCGCUUAUUUAUCGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2125 -1:5 GCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAA AGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2126 -83:87 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAGAUAAAUAAGAAGCAUCAAAG 2127 =+G28, A82T, UACUGGCGCUUUUAUCUCAUUACUUUGGAGAGCCAUCACCAGCGACUAUGUCGUAU -84. GGGUAAAGCGCUUAUUUAUCGGAGAGUAUCCGAUAAAUAAGAAGCAUCAAAG 2128 =+51T UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUUCGUAU GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2129 -1:4, +G5A, AGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUA +G86, AAGCGCUUAUUUAUCGGAGAGAAAUGCCGAUAAAUAAGAAGCAUCAAAG 2130 =+A94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAUAAGAAGCAUCAAAG 2131 =+G72 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUGUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2132 shorten front, GCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAA CUUCGG loop AGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGCGCAUCAAAG modified, extend extended 2133 A14C UACUGGCGCUUUUCUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUUAAUAAGAAGCAUCAAAG 2134 -1:3, +G3 GUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGG UAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2135 =+C45, +T46 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACCUUAUGUCGUA UGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2136 CUUCGG loop GAUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG modified, fun GUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG start 2137 -93:94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAGAAGCAUCAAAG 2138 =+T45 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGAUCUAUGUCGUAU GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2139 -69, -94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAAGAAGCAUCAAAG 2140 -94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAAGAAGCAUCAAAG 2141 modified UACUGGCGCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG CUUCGG, GUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG minus T in 1st triplex 2142 -1:4, +C4, A14C, CGGCGCUUUUCUCGCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGU T17G, +G72, AAAGCGCUUAUUGUAUCGAGAGAUAAAUAAGAAGCAUCAAAG -76:78, -83:87 2143 T1C, -73 CACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2144 Scaffold uuCG, UACUGGCGCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUG stem uuCG. Stem GGUAAAGCGCUUAUGUAUCGGCUUCGGCCGAUACAUAAGAAGCAUCAAAG swap, t shorten 2145 Scaffold uuCG, UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAU stem uuCG. Stem GGGUAAAGCGCUUAUGUAUCGGCUUCGGCCGAUACAUAAGAAGCAUCAAAG swap 2146 =+G60 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUGAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2147 no stem Scaffold UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAU uuCG GGGUAAAG 2148 no stem Scaffold GAUGGGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUGG uuCG, fun start GUAAAG 2149 Scaffold uuCG, GAUGGGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUGG stem uuCG, fun GUAAAGCGCUUAUUUAUCGGCUUCGGCCGAUAAAUAAGAAGCAUCAAAG start 2150 Pseudoknots UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUA UACUUUGGAGUUUUAAAAUGUCUCUAAGUACAGAAGCAUCAAAG 2151 Scaffold uuCG, GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUGGGU stem uuCG AAAGCGCUUAUUUAUCGGCUUCGGCCGAUAAAUAAGAAGCAUCAAAG 2152 Scaffold uuCG, GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUG stem uuCG, no GGUAAAGCGCUUAUUUAUCGGCUUCGGCCGAUAAAUAAGAAGCAUCAAAG start 2153 Scaffold uuCG UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAU GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2154 =+GCTC36 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUGCUCCACCAGCGACUAUGUCG UAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2155 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG telomere basket + GGUAAAGCGGGGUUAGGGUUAGGGUUAGGGAAGCAUCAAAG ends 2156 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG M3q GGUAAAGCGGAGGGAGGGAGGGAGAGGGAAAGCAUCAAAG 2157 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG telomere basket GGUAAAGCGUUGGGUUAGGGUUAGGGUUAGGGAAAAGCAUCAAAG no ends 2158 45, 44 hairpin UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG (old version) GGUAAAGCGC--------AGGGCUUCGGCCG---------GAAGCAUCAAAG 2159 Sarcin-ricin loop UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCCUGCUCAGUACGAGAGGAACCGCAGGAAGCAUCAAAG 2160 uvsX, C18G UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG 2161 truncated stem UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG loop, C18G, trip GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG mut (T10C) 2162 short phage rep, UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG C18G GGUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG 2163 phage rep loop, UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG C18G GGUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG 2164 =+G18, stacked UACUGGCGCCUUUAUCUGCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU onto 64 GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2165 truncated stem GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG loop, C18G, -1 GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG A2G 2166 phage rep loop, UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG C18G, trip mut GGUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG (T10C) 2167 short phage rep, UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG C18G, trip mut GGUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG (T10C) 2168 uvsX, trip mut UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG (T10C) GGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG 2169 truncated stem UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG loop GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2170 =+A17, stacked UACUGGCGCCUUUAUCAUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU onto 64 GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2171 3′ HDV genomic UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG ribozyme GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGCC GGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGACCGU CCCCUCGGUAAUGGCGAAUGGGACCC 2172 hage rep loop, UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG trip mut (T10C) GGUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG 2173 -79:80 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2174 short phage rep, UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG trip mut (T10C) GGUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG 2175 extra truncated UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG stem loop GGUAAAGCGCCGGACUUCGGUCCGGAAGCAUCAAAG 2176 T17G, C18G UACUGGCGCUUUUAUCGGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2177 short phage rep UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG 2178 uvsX, C18G, -1 GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG A2G GUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG 2179 uvsX, C18G, trip GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG mut (T10C), -1 GUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG A2G, HDV -99 G65U 2180 3′ HDV UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG antigenomic GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGGU ribozyme CGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGACGCA CGUCCACUCGGAUGGCUAAGGGAGAGCCA 2181 uvsX, C18G, trip GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG mut (T10C), -1 GUAAAGCGCCCUCUUCGGAGGGCGCAUCAAAG A2G, HDV AA(98:99)C 2182 3′ HDV ribozyme UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG (Lior Nissim, GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGUUUU Timothy Lu) GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAU GGCGAAUGGGACCCCGGG 2183 TAC(1:3)GA, GAUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG stacked onto 64 GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2184 uvsX, -1 A2G GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG GUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG 2185 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG loop, C18G, trip GUAAAGCUCUUACGGACUUCGGUCCGUAAGAGCAUCAAAG mut (T10C), -1 A2G, HDV -99 G65U 2186 short phage rep, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG C18G, trip mut GUAAAGCUCGGACGACCUCUCGGUCGUCCGAGCAUCAAAG (T10C), -1 A2G, HDV -99 G65U 2187 3′ sTRSV WT UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG viral GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCCUG Hammerhead UCACCGGAUGUGCUUUCCGGUCUGAUGAGUCCGUGAGGACGAAACAGG ribozyme 2188 short phage rep, GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG C18G, -1 A2G GUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG 2189 short phage rep, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG C18G, trip mut GUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG (T10C), -1 A2G, 3′ genomic HDV 2190 phage rep loop, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG C18G, trip mut GUAAAGCUCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAGCAUCAAAG (T10C), -1 A2G, HDV -99 G65U 2191 3′ HDV ribozyme UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG (Owen Ryan, GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGAUG Jamie Cate) GCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACACCUUCGGGUGGC GAAUGGGAC 2192 phage rep loop, GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG C18G, -1 A2G GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG 2193 0.14 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUACU GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2194 -78, G77T UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGUGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2195 GUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2196 short phage rep, GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG -1 A2G GUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG 2197 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG loop, C18G, trip GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG mut (T10C), -1 A2G 2198 -1, A2G GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG GUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2199 truncated stem GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG loop, trip mut GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG (T10C), -1 A2G 2200 uvsX, C18G, trip GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG mut (T10C), -1 GUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG A2G 2201 phage rep loop, GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG -1 A2G GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG 2202 phage rep loop, GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG trip mut (T10C), GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG -1 A2G 2203 phage rep loop, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG C18G, trip mut GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG (T10C), -1 A2G 2204 truncated stem UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG loop, C18G GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2205 uvsX, trip mut GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG (T10C), -1 A2G GUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG 2206 truncated stem GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG loop, -1 A2G GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2207 short phage rep, GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG trip mut (T10C), GUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG -1 A2G 2208 5′ HDV ribozyme GAUGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACACCUUCGGG (Owen Ryan, UGGCGAAUGGGACUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCG Jamie Cate) ACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAA GCAUCAAAG 2209 5′ HDV genomic GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGA ribozyme CCGUCCCCUCGGUAAUGGCGAAUGGGACCCUACUGGCGCUUUUAUCUCAUUACUUU GAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGA AAUCCGAUAAAUAAGAAGCAUCAAAG 2210 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG loop, C18G, trip GUAAAGCGCUUACGGACUUCGGUCCGUAAGCGCAUCAAAG mut (T10C), -1 A2G, HDV AA(98:99)C 2211 5′ env25 pistol CGUGGUUAGGGCCACGUUAAAUAGUUGCUUAAGCCCUAAGCGUUGAUCUUCGGAUC ribozyme (with AGGUGCAAUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAU an added GUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUC CUUCGG loop) AAAG 2212 5′ HDV GGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGA antigenomic CGCACGUCCACUCGGAUGGCUAAGGGAGAGCCAUACUGGCGCUUUUAUCUCAUUAC ribozyme UUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAG AGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2213 3′ Hammerhead UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG ribozyme (Lior GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCCAG Nissim, Timothy UACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUACUGGCGCUUUUAUCU Lu) guide CAU scaffold scar 2214 =+A27, stacked UACUGGCGCCUUUAUCUCAUUACUUUAGAGAGCCAUCACCAGCGACUAUGUCGUAU onto 64 GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2215 5′ Hammerhead CGACUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCGUACUGGC ribozyme (Lior GCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAG Nissim, Timothy CGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG Lu) smaller scar 2216 phage rep loop, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG C18G, trip mut GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGCGCAUCAAAG (T10C), -1 A2G, HDV AA(98:99)C 2217 -27, stacked onto UACUGGCGCCUUUAUCUCAUUACUUUAGAGCCAUCACCAGCGACUAUGUCGUAUGG 64 GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2218 3′ Hatchet UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCAUU CCUCAGAAAAUGACAAACCUGUGGGGCGUAAGUAGAUCUUCGGAUCUAUGAUCGUG CAGACGUUAAAAUCAGGU 2219 3′ Hammerhead UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG ribozyme (Lior GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGAC Nissim, Timothy UACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCGCGUGUAGCGAA Lu) GCA 2220 5′ Hatchet CAUUCCUCAGAAAAUGACAAACCUGUGGGGCGUAAGUAGAUCUUCGGAUCUAUGAU CGUGCAGACGUUAAAAUCAGGUUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCA UCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAU AAAUAAGAAGCAUCAAAG 2221 5′ HDV ribozyme UUUUGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCG (Lior Nissim, GCAUGGCGAAUGGGACCCCGGGUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCA Timothy Lu) UCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAU AAAUAAGAAGCAUCAAAG 2222 5′ Hammerhead CGACUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCGCGUGUAG ribozyme (Lior CGAAGCAUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUG Nissim, Timothy UCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCA Lu) AAG 2223 3′ HH15 Minimal UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG Hammerhead GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGGA ribozyme GCCCCGCUGAUGAGGUCGGGGAGACCGAAAGGGACUUCGGUCCCUACGGGGCUCCC 2224 5′ RBMX CCACCCCCACCACCACCCCCACCCCCACCACCACCCUACUGGCGCUUUUAUCUCAU recruiting motif UACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCG GAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2225 3′ Hammerhead UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG ribozyme (Lior GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGAC Nissim, Timothy UACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCG Lu) smaller scar 2226 3′ env25 pistol UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG ribozyme (with GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGUG an added GUUAGGGCCACGUUAAAUAGUUGCUUAAGCCCUAAGCGUUGAUCUUCGGAUCAGGU CUUCGG loop) GCAA 2227 3′ Env-9 Twister UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGCA AUAAAGCGGUUACAAGCCCGCAAAAAUAGCAGAGUAAUGUCGCGAUAGCGCGGCAU UAAUGCAGCUUUAUUG 2228 =+ATTATCTCA UACUGGCGCUUUUAUCUCAUUACUAUUAUCUCAUUACUUUGAGAGCCAUCACCAGC TTACT25 GACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGA AGCAUCAAAG 2229 5′ Env-9 Twister GGCAAUAAAGCGGUUACAAGCCCGCAAAAAUAGCAGAGUAAUGUCGCGAUAGCGCG GCAUUAAUGCAGCUUUAUUGUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUC ACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAA AUAAGAAGCAUCAAAG 2230 3′ Twisted Sister UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG 1 GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGACCC GCAAGGCCGACGGCAUCCGCCGCCGCUGGUGCAAGUCCAGCCGCCCCUUCGGGGGC GGGCGCUCAUGGGUAAC 2231 no stem UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG GGUAAAG 2232 5′ HH15 Minimal GGGAGCCCCGCUGAUGAGGUCGGGGAGACCGAAAGGGACUUCGGUCCCUACGGGGC Hammerhead UCCCUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCG ribozyme UAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 2233 5′ Hammerhead CCAGUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUACUGGCGCUUUU ribozyme (Lior AUCUCAUUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUG Nissim, Timothy UCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCA Lu) guide AAG scaffold scar 2234 5′ Twisted Sister ACCCGCAAGGCCGACGGCAUCCGCCGCCGCUGGUGCAAGUCCAGCCGCCCCUUCGG 1 GGGCGGGCGCUCAUGGGUAACUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAU CACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUA AAUAAGAAGCAUCAAAG 2235 5′ sTRSV WT CCUGUCACCGGAUGUGCUUUCCGGUCUGAUGAGUCCGUGAGGACGAAACAGGUACU viral GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUA Hammerhead AAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG ribozyme 2236 148, =+G55, GUACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG stacked onto 64 UGGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2237 158, GUACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG 103 + 148(+G55) UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG -99, G65U 2238 174, Uvsx ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG Extended stem GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG with [A99] G65U), C18G, {circumflex over ( )}G55, [GT-1] 2239 175, extended ACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG stem truncation, GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG T10C, [GT-1] 2240 176, 174 with GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG A1G substitution GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG for T7 transcription 2241 177, 174 with ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG bubble (+G55) GUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG removed 2242 181, stem 42 ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG (truncated stem GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG loop); T10C, C18G, [GT -1](95 + [GT-1]) 2243 182, stem 42 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG (truncated stem GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG loop); C18G, [GT-1] 2244 183, stem 42 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG (truncated stem GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG loop); C18G, {circumflex over ( )}G55, [GT- 1] 2245 184, stem 48 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUUG (uvsx, -99 g65t); GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG C18G, {circumflex over ( )}T55, [GT- 1] 2246 185, stem 42 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUUG (truncated stem GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG loop); C18G, {circumflex over ( )}T55, [GT- 1] 2247 186, stem 42 ACUGGCGCCUUUAUCAUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG (truncated stem GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG loop); T10C, {circumflex over ( )}A17, [GT- 1] 2248 187, stem 46 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG (uvsx); GGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG C18G, {circumflex over ( )}G55, [GT- 1] 2249 188, stem 50 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG (ms2 U15C, -99, GGUAAAGCUCACAUGAGGAUCACCCAUGUGAGCAUCAAAG g65t); C18G, {circumflex over ( )}G55, [GT- 1] 2250 189, 174 + ACUGGCACUUUUACCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAGUG G8A; T15C; T35A GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2251 190, 174 + G8A ACUGGCACUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2252 191, 174 + G8C ACUGGCCCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2253 192, 174 + T15C ACUGGCGCUUUUACCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2254 193, 174 + T35A ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2255 195, 175 + C18G + ACUGGCACCUUUACCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAUGG G8A; T15C; T35A GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2256 196, 175 + C18G + ACUGGCACCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG G8A GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2257 197, 175 + C18G + ACUGGCCCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG G8C GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2258 198, 175 + C18G + ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAUGG T35A GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2259 199, 174 + A2G GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG (test G GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG transcription at start; ccGCT...) 2260 200, 174 + {circumflex over ( )}G1 GACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGU (ccGACT...) GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2261 201, 174 + ACUGGCGCCUUUAUCUGAUUACUUUGGAGAGCCAUCACCAGCGACUAUGUCGUAGU T10C; {circumflex over ( )}G28 GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2262 202, 174 + ACUGGCGCAUUUAUCUGAUUACUUUGUGAGCCAUCACCAGCGACUAUGUCGUAGUG T10A; A28T GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2263 203, 174 + T10C ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2264 204, 174 + {circumflex over ( )}G28 ACUGGCGCUUUUAUCUGAUUACUUUGGAGAGCCAUCACCAGCGACUAUGUCGUAGU GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2265 205, 174 + T10A ACUGGCGCAUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2266 206, 174 + A28T ACUGGCGCUUUUAUCUGAUUACUUUGUGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2267 207, 174 + {circumflex over ( )}T15 ACUGGCGCUUUUAUUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGU GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2268 208, 174 + [T4] ACGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGG GUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2269 209, 174 + C16A ACUGGCGCUUUUAUAUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2270 210, 174 + {circumflex over ( )}T17 ACUGGCGCUUUUAUCUUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGU GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2271 211, 174 + T35G ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAGCACCAGCGACUAUGUCGUAGUG (compare with GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 174 + T35A above) 2272 212, 174 + U11G, ACUGGCGCUGUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAGUG A105G (A86G), GGUAAAGCUCCCUCUUCGGAGGGAGCAUCGAAG U26C 2273 213, 174 + U11C, ACUGGCGCUCUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAGUG A105G (A86G), GGUAAAGCUCCCUCUUCGGAGGGAGCAUCGAAG U26C 2274 214, 174 + U12G; ACUGGCGCUUGUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG A106G (A87G), GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG U25C 2275 215, 174 + U12C; ACUGGCGCUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG A106G (A87G), GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG U25C 2276 216, ACUGGCGCUUUGAUCUGAUUACCUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG 174_tx_tt.G,87. GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAGG G,22.C 2277 217, ACUGGCGCUUUCAUCUGAUUACCUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG 174_tx_11.C,87. GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAGG G,22.C 2278 218, 174 + U11G ACUGGCGCUGUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2279 219, 174 + ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG A105G (A86G) GGUAAAGCUCCCUCUUCGGAGGGAGCAUCGAAG 2280 220, 174 + U26C ACUGGCGCUUUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAGUG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

In some embodiments, the gNA variant comprises a tracrRNA stem loop comprising the sequence -UUU-N4-25-UUU- (SEQ ID NO: 240). For example, the gNA variant comprises a scaffold stem loop or a replacement thereof, flanked by two triplet U motifs that contribute to the triplex region. In some embodiments, the scaffold stem loop or replacement thereof comprises at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides.

In some embodiments, the gNA variant comprises a crRNA sequence with -AAAG- in a location 5′ to the spacer region. In some embodiments, the -AAAG- sequence is immediately 5′ to the spacer region.

In some embodiments, the at least one nucleotide modification to a reference gNA to produce a gNA variant comprises at least one nucleotide deletion in the CasX variant gNA relative to the reference gRNA. In some embodiments, a gNA variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive or non-consecutive nucleotides relative to a reference gNA. In some embodiments, the at least one deletion comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gNA. In some embodiments, the gNA variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotide deletions relative to the reference gNA, and the deletions are not in consecutive nucleotides. In those embodiments where there are two or more non-consecutive deletions in the gNA variant relative to the reference gRNA, any length of deletions, and any combination of lengths of deletions, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first deletion of one nucleotide, and a second deletion of two nucleotides and the two deletions are not consecutive. In some embodiments, a gNA variant comprises at least two deletions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two deletions in the same region of the reference gRNA. For example, the regions may be the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5′ end of the gNA variant. The deletion of any nucleotide in a reference gRNA is contemplated as within the scope of the disclosure.

In some embodiments, the at least one nucleotide modification of a reference gRNA to generate a gNA variant comprises at least one nucleotide insertion. In some embodiments, a gNA variant comprises an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive or non-consecutive nucleotides relative to a reference gRNA. In some embodiments, the at least one nucleotide insertion comprises an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gRNA. In some embodiments, the gNA variant comprises 2 or more insertions relative to the reference gRNA, and the insertions are not consecutive. In those embodiments where there are two or more non-consecutive insertions in the gNA variant relative to the reference gRNA, any length of insertions, and any combination of lengths of insertions, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first insertion of one nucleotide, and a second insertion of two nucleotides and the two insertions are not consecutive. In some embodiments, a gNA variant comprises at least two insertions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two insertions in the same region of the reference gRNA. For example, the regions may be the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5′ end of the gNA variant. Any insertion of A, G, C, U (or T, in the corresponding DNA) or combinations thereof at any location in the reference gRNA is contemplated as within the scope of the disclosure.

In some embodiments, the at least one nucleotide modification of a reference gRNA to generate a gNA variant comprises at least one nucleic acid substitution. In some embodiments, a gNA variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive or non-consecutive substituted nucleotides relative to a reference gRNA. In some embodiments, a gNA variant comprises 1-4 nucleotide substitutions relative to a reference gRNA. In some embodiments, the at least one substitution comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gRNA. In some embodiments, the gNA variant comprises 2 or more substitutions relative to the reference gRNA, and the substitutions are not consecutive. In those embodiments where there are two or more non-consecutive substitutions in the gNA variant relative to the reference gRNA, any length of substituted nucleotides, and any combination of lengths of substituted nucleotides, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first substitution of one nucleotide, and a second substitution of two nucleotides and the two substitutions are not consecutive. In some embodiments, a gNA variant comprises at least two substitutions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two substitutions in the same region of the reference gRNA. For example, the regions may be the triplex, the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5′ end of the gNA variant. Any substitution of A, G, C, U (or T, in the corresponding DNA) or combinations thereof at any location in the reference gRNA is contemplated as within the scope of the disclosure.

Any of the substitutions, insertions and deletions described herein can be combined to generate a gNA variant of the disclosure. For example, a gNA variant can comprise at least one substitution and at least one deletion relative to a reference gRNA, at least one substitution and at least one insertion relative to a reference gRNA, at least one insertion and at least one deletion relative to a reference gRNA, or at least one substitution, one insertion and one deletion relative to a reference gRNA.

In some embodiments, the gNA variant comprises a scaffold region at least 20% identical, at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any one of SEQ ID NOS: 4-16. In some embodiments, the gNA variant comprises a scaffold region at least 60% homologous (or identical) to any one of SEQ ID NOS: 4-16.

In some embodiments, the gNA variant comprises a tracr stem loop at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 14. In some embodiments, the gNA variant comprises a tracr stem loop at least 60% homologous (or identical) to SEQ ID NO: 14.

In some embodiments, the gNA variant comprises an extended stem loop at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 15. In some embodiments, the gNA variant comprises an extended stem loop at least 60% homologous (or identical) to SEQ ID NO: 15.

In some embodiments, a gNA variant comprises a sequence of any one of SEQ ID NOs: 412-3295. In some embodiments, a gNA variant comprises a sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280. In some embodiments, a gNA variant comprises a sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

In some embodiments, the gNA variant comprises an exogenous extended stem loop, with such differences from a reference gNA described as follows. In some embodiments, an exogenous extended stem loop has little or no identity to the reference stem loop regions disclosed herein (e.g., SEQ ID NO: 15). In some embodiments, an exogenous stem loop is at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1,000 bp, at least 2,000 bp, at least 3,000 bp, at least 4,000 bp, at least 5,000 bp, at least 6,000 bp, at least 7,000 bp, at least 8,000 bp, at least 9,000 bp, at least 10,000 bp, at least 12,000 bp, at least 15,000 bp or at least 20,000 bp. In some embodiments, the gNA variant comprises an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides. In some embodiments, the heterologous stem loop increases the stability of the gNA. In some embodiments, the heterologous RNA stem loop is capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule. In some embodiments, an exogenous stem loop region comprises an RNA stem loop or hairpin, for example a thermostable RNA such as MS2 (ACAUGAGGAUUACCCAUGU; SEQ ID NO: 4278), Qβ (UGCAUGUCUAAGACAGCA; SEQ ID NO: 4279), U1 hairpin II (AAUCCAUUGCACUCCGGAUU; SEQ ID NO:4280), Uvsx (CCUCUUCGGAGG; SEQ ID NO: 4281), PP7 (AGGAGUUUCUAUGGAAACCCU; SEQ ID NO: 4282), Phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU; SEQ ID NO: 4283), Kissing loop_a (UGCUCGCUCCGUUCGAGCA; SEQ ID NO: 4284), Kissing loop_b1 (UGCUCGACGCGUCCUCGAGCA; SEQ ID NO: 4285), Kissing loop_b2 (UGCUCGUUUGCGGCUACGAGCA; SEQ ID NO: 4286), G quadriplex M3q (AGGGAGGGAGGGAGAGG; SEQ ID NO: 4287), G quadriplex telomere basket (GGUUAGGGUUAGGGUUAGG; SEQ ID NO: 4288), Sarcin-ricin loop (CUGCUCAGUACGAGAGGAACCGCAG; SEQ ID NO: 4289) or Pseudoknots (UACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUUUGG AGUUUUAAAAUGUCUCUAAGUACA; SEQ ID NO: 4290). In some embodiments, an exogenous stem loop comprises an RNA scaffold. As used herein, an “RNA scaffold” refers to a multi-dimensional RNA structure capable of interacting with and organizing or localizing one or more proteins. In some embodiments, the RNA scaffold is synthetic or non-naturally occurring. In some embodiments, an exogenous stem loop comprises a long non-coding RNA (lncRNA). As used herein, a lncRNA refers to a non-coding RNA that is longer than approximately 200 bp in length. In some embodiments, the 5′ and 3′ ends of the exogenous stem loop are base paired, i.e., interact to form a region of duplex RNA. In some embodiments, the 5′ and 3′ ends of the exogenous stem loop are base paired, and one or more regions between the 5′ and 3′ ends of the exogenous stem loop are not base paired. In some embodiments, the at least one nucleotide modification comprises: (a) substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (b) a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (c) an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (d) a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5′ and 3′ ends; or any combination of (a)-(d).

In some embodiments, a gNA variant comprises a sequence or subsequence of any one of SEQ ID NOs: 412-3295 and an a sequence of an exogenous stem loop. In some embodiments, a gNA variant comprises a sequence or subsequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280 and a sequence of an exogenous stem loop. In some embodiments, a gNA variant comprises a sequence or subsequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280 and a sequence of an exogenous stem loop.

In some embodiments, the gNA variant comprises a scaffold stem loop having at least 60% identity to SEQ ID NO: 14. In some embodiments, the gNA variant comprises a scaffold stem loop having at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity or at least 99% identity to SEQ ID NO: 14. In some embodiments, the gNA variant comprises a scaffold stem loop comprising SEQ ID NO: 14.

In some embodiments, the gNA variant comprises a scaffold stem loop sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 245). In some embodiments, the gNA variant comprises a scaffold stem loop sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 245) with at least 1, 2, 3, 4, or 5 mismatches thereto.

In some embodiments, the gNA variant comprises an extended stem loop region comprising less than 32 nucleotides, less than 31 nucleotides, less than 30 nucleotides, less than 29 nucleotides, less than 28 nucleotides, less than 27 nucleotides, less than 26 nucleotides, less than 25 nucleotides, less than 24 nucleotides, less than 23 nucleotides, less than 22 nucleotides, less than 21 nucleotides, or less than 20 nucleotides. In some embodiments, the gNA variant comprises an extended stem loop region comprising less than 32 nucleotides. In some embodiments, the gNA variant further comprises a thermostable stem loop.

In some embodiments, a sgRNA variant comprises a sequence of SEQ ID NO: 2104, 2106, SEQ ID NO: 2163, SEQ ID NO: 2107, SEQ ID NO: 2164, SEQ ID NO: 2165, SEQ ID NO: 2166, SEQ ID NO: 2103, SEQ ID NO: 2167, SEQ ID NO: 2105, SEQ ID NO: 2108, SEQ ID NO: 2112, SEQ ID NO: 2160, SEQ ID NO: 2170, SEQ ID NO: 2114, SEQ ID NO: 2171, SEQ ID NO: 2112, SEQ ID NO: 2173, SEQ ID NO: 2102, SEQ ID NO: 2174, SEQ ID NO: 2175, SEQ ID NO: 2109, SEQ ID NO: 2176, SEQ ID NO: 2238, SEQ ID NO: 2239, SEQ ID NO: 2240, or SEQ ID NO: 2241.

In some embodiments, the gNA variant comprises one or more additional changes to a sequence of any one of SEQ ID NOs: 2201-2280. In some embodiments, the gNA variant comprises a sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280, or having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98/6, at least about 99% identity thereto. In some embodiments, the gNA variant comprises one or more additional changes to a sequence of any one of SEQ ID NOs: 2201-2280. In some embodiments, the gNA variant comprises the sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

In some embodiments, a sgRNA variant comprises one or more additional changes to a sequence of SEQ ID NO: 2104, SEQ ID NO: 2163, SEQ ID NO: 2107, SEQ ID NO: 2164, SEQ ID NO: 2165, SEQ ID NO: 2166, SEQ ID NO: 2103, SEQ ID NO: 2167, SEQ ID NO: 2105, SEQ ID NO: 2108, SEQ ID NO: 2112, SEQ ID NO: 2160, SEQ ID NO: 2170, SEQ ID NO: 2114, SEQ ID NO: 2171, SEQ ID NO: 2112, SEQ ID NO: 2173, SEQ ID NO: 2102, SEQ ID NO: 2174, SEQ ID NO: 2175, SEQ ID NO: 2109, SEQ ID NO: 2176, SEQ ID NO: 2238, SEQ ID NO: 2239, SEQ ID NO: 2240, or SEQ ID NO: 2241.

In some embodiments of the gNA variants of the disclosure, the gNA variant comprises at least one modification, wherein the at least one modification compared to the reference guide scaffold of SEQ ID NO: 5 is selected from one or more of: (a) a C18G substitution in the triplex loop; (b) a G55 insertion in the stem bubble; (c) a U1 deletion; (d) a modification of the extended stem loop wherein (i) a 6 nt loop and 13 loop-proximal base pairs are replaced by a Uvsx hairpin; and (ii) a deletion of A99 and a substitution of G65U that results in a loop-distal base that is fully base-paired. In such embodiments, the gNA variant comprises the sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

In some embodiments, the scaffold of the gNA variant comprises the sequence of any one of SEQ ID NOS: 2201-2280 of Table 2. In some embodiments, the scaffold of the gNA consists or consists essentially of the sequence of any one of SEQ ID NOS: 2201-2280. In some embodiments, the scaffold of the gNA variant sequence is at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical or at least about 99% identical to any one of SEQ ID NOS: 2201 to 2280.

In some embodiments, the gNA variant further comprises a spacer (or targeting sequence) region, described more fully, supra, which comprises at least 14 to about 35 nucleotides wherein the spacer is designed with a sequence that is complementary to a target DNA. In some embodiments, the gNA variant comprises a targeting sequence of at least 10 to 30 nucleotides complementary to a target DNA. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides. In some embodiments, the gNA variant comprises a targeting sequence having 20 nucleotides. In some embodiments, the targeting sequence has 25 nucleotides. In some embodiments, the targeting sequence has 24 nucleotides. In some embodiments, the targeting sequence has 23 nucleotides. In some embodiments, the targeting sequence has 22 nucleotides. In some embodiments, the targeting sequence has 21 nucleotides. In some embodiments, the targeting sequence has 20 nucleotides. In some embodiments, the targeting sequence has 19 nucleotides. In some embodiments, the targeting sequence has 18 nucleotides. In some embodiments, the targeting sequence has 17 nucleotides. In some embodiments, the targeting sequence has 16 nucleotides. In some embodiments, the targeting sequence has 15 nucleotides. In some embodiments, the targeting sequence has 14 nucleotides.

In some embodiments, the scaffold of the gNA variant is a variant comprising one or more additional changes to a sequence of a reference gRNA that comprises SEQ ID NO: 4 or SEQ ID NO: 5. In those embodiments where the scaffold of the reference gRNA is derived from SEQ ID NO: 4 or SEQ ID NO: 5, the one or more improved or added characteristics of the gNA variant are improved compared to the same characteristic in SEQ ID NO: 4 or SEQ ID NO: 5.

In some embodiments, the scaffold of the gNA variant is part of an RNP with a reference CasX protein comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In other embodiments, the scaffold of the gNA variant is part of an RNP with a CasX variant protein comprising any one of the sequences of Tables 3, 8, 9, 10 and 12, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In the foregoing embodiments, the gNA further comprises a spacer sequence.

h. Chemically Modified gNAs

In some embodiments, the disclosure provides chemically-modified gNAs. In some embodiments, the present disclosure provides a chemically-modified gNA that has guide NA functionality and has reduced susceptibility to cleavage by a nuclease. A gNA that comprises any nucleotide other than the four canonical ribonucleotides A, C, G, and U, or a deoxynucleotide, is a chemically modified gNA. In some cases, a chemically-modified gNA comprises any backbone or internucleotide linkage other than a natural phosphodiester internucleotide linkage. In certain embodiments, the retained functionality includes the ability of the modified gNA to bind to a CasX of any of the embodiments described herein. In certain embodiments, the retained functionality includes the ability of the modified gNA to bind to a target nucleic acid sequence. In certain embodiments, the retained functionality includes targeting a CasX protein or the ability of a pre-complexed RNP to bind to a target nucleic acid sequence. In certain embodiments, the retained functionality includes the ability to nick a target polynucleotide by a CasX-gNA. In certain embodiments, the retained functionality includes the ability to cleave a target nucleic acid sequence by a CasX-gNA. In certain embodiments, the retained functionality is any other known function of a gNA in a recombinant system with a CasX chimera protein of the embodiments of the disclosure.

In some embodiments, the disclosure provides a chemically-modified gNA in which a nucleotide sugar modification is incorporated into the gNA selected from the group consisting of 2′-O—C₁₋₄alkyl such as 2′-O-methyl (2′-OMe), 2′-deoxy (2′-H), 2′-O—C₁₋₃alkyl-O—C₁₋₃alkyl such as 2′-methoxyethyl (“2′-MOE”), 2′-fluoro (“2′-F”), 2′-amino (“2′-NH₂”), 2′-arabinosyl (“2′-arabino”) nucleotide, 2′-F-arabinosyl (“2′-F-arabino”) nucleotide, 2′-locked nucleic acid (“LNA”) nucleotide, 2′-unlocked nucleic acid (“ULNA”) nucleotide, a sugar in L form (“L-sugar”), and 4′-thioribosyl nucleotide. In other embodiments, an internucleotide linkage modification incorporated into the guide RNA is selected from the group consisting of: phosphorothioate “P(S)” (P(S)), phosphonocarboxylate (P(CH₂)_(n)COOR) such as phosphonoacetate “PACE” (P(CH₂COO⁻)), thiophosphonocarboxylate ((S)P(CH₂)_(n)COOR) such as thiophosphonoacetate “thioPACE” ((S)P(CH₂)_(n)COO⁻)), alkylphosphonate (P(C₁₋₃alkyl) such as methylphosphonate —P(CH₃), boranophosphonate (P(BH₃)), and phosphorodithioate (P(S)₂).

In certain embodiments, the disclosure provides a chemically-modified gNA in which a nucleobase (“base”) modification is incorporated into the gNA selected from the group consisting of: 2-thiouracil (“2-thioU”), 2-thiocytosine (“2-thioC”), 4-thiouracil (“4-thioU”), 6-thioguanine (“6-thioG”), 2-aminoadenine (“2-aminoA”), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine (“5-methylC”), 5-methyluracil (“5-methylU”), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil (“5-allylU”), 5-allylcytosine (“5-allylC”), 5-aminoallyluracil (“5-aminoallylU”), 5-aminoallyl-cytosine (“5-aminoallylC”), an abasic nucleotide, Z base, P base, Unstructured Nucleic Acid (“UNA”), isoguanine (“isoG”), isocytosine (“isoC”), 5-methyl-2-pyrimidine, x(A,G,C,T) and y(A,G,C,T).

In other embodiments, the disclosure provides a chemically-modified gNA in which one or more isotopic modifications are introduced on the nucleotide sugar, the nucleobase, the phosphodiester linkage and/or the nucleotide phosphates, including nucleotides comprising one or more ¹⁵N, ¹³C, ¹⁴C, deuterium, ³H, ³²P, ¹²⁵I, ¹³¹I atoms or other atoms or elements used as tracers.

In some embodiments, an “end” modification incorporated into the gNA is selected from the group consisting of: PEG (polyethyleneglycol), hydrocarbon linkers (including: heteroatom (O,S,N)-substituted hydrocarbon spacers; halo-substituted hydrocarbon spacers; keto-, carboxyl-, amido-, thionyl-, carbamoyl-, thionocarbamaoyl-containing hydrocarbon spacers), spermine linkers, dyes including fluorescent dyes (for example fluoresceins, rhodamines, cyanines) attached to linkers such as, for example 6-fluorescein-hexyl, quenchers (for example dabcyl, BHQ) and other labels (for example biotin, digoxigenin, acridine, streptavidin, avidin, peptides and/or proteins). In some embodiments, an “end” modification comprises a conjugation (or ligation) of the gNA to another molecule comprising an oligonucleotide of deoxynucleotides and/or ribonucleotides, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin and/or other molecule. In certain embodiments, the disclosure provides a chemically-modified gNA in which an “end” modification (described above) is located internally in the gNA sequence via a linker such as, for example, a 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker, which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the gNA.

In some embodiments, the disclosure provides a chemically-modified gNA having an end modification comprising a terminal functional group such as an amine, a thiol (or sulfhydryl), a hydroxyl, a carboxyl, carbonyl, thionyl, thiocarbonyl, a carbamoyl, a thiocarbamoyl, a phosphoryl, an alkene, an alkyne, an halogen or a functional group-terminated linker that can be subsequently conjugated to a desired moiety selected from the group consisting of a fluorescent dye, a non-fluorescent label, a tag (for ¹⁴C, example biotin, avidin, streptavidin, or moiety containing an isotopic label such as ¹³N, ¹³C, deuterium, ³H, ³²P, ¹²⁵I and the like), an oligonucleotide (comprising deoxynucleotides and/or ribonucleotides, including an aptamer), an amino acid, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, and a vitamin. The conjugation employs standard chemistry well-known in the art, including but not limited to coupling via N-hydroxysuccinimide, isothiocyanate, DCC (or DCI), and/or any other standard method as described in “Bioconjugate Techniques” by Greg T. Hermanson, Publisher Elsevier Science, 3^(rd) ed. (2013), the contents of which are incorporated herein by reference in its entirety.

i. Complex Formation with CasX Protein

In some embodiments, a gNA variant has an improved ability to form a complex with a CasX protein (such as a reference CasX or a CasX variant protein) when compared to a reference gRNA. In some embodiments, a gNA variant has an improved affinity for a CasX protein (such as a reference or variant protein) when compared to a reference gRNA, thereby improving its ability to form a ribonucleoprotein (RNP) complex with the CasX protein, as described in the Examples. Improving ribonucleoprotein complex formation may, in some embodiments, improve the efficiency with which functional RNPs are assembled. In some embodiments, greater than 90%, greater than 93%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% of RNPs comprising a gNA variant and a spacer are competent for gene editing of a target nucleic acid.

Exemplary nucleotide changes that can improve the ability of gNA variants to form a complex with CasX protein may, in some embodiments, include replacing the scaffold stem with a thermostable stem loop. Without wishing to be bound by any theory, replacing the scaffold stem with a thermostable stem loop could increase the overall binding stability of the gNA variant with the CasX protein. Alternatively, or in addition, removing a large section of the stem loop could change the gNA variant folding kinetics and make a functional folded gNA easier and quicker to structurally-assemble, for example by lessening the degree to which the gNA variant can get “tangled” in itself. In some embodiments, choice of scaffold stem loop sequence could change with different spacers that are utilized for the gNA. In some embodiments, scaffold sequence can be tailored to the spacer and therefore the target sequence. Biochemical assays can be used to evaluate the binding affinity of CasX protein for the gNA variant to form the RNP, including the assays of the Examples. For example, a person of ordinary skill can measure changes in the amount of a fluorescently tagged gNA that is bound to an immobilized CasX protein, as a response to increasing concentrations of an additional unlabeled “cold competitor” gNA. Alternatively, or in addition, fluorescence signal can be monitored to or seeing how it changes as different amounts of fluorescently labeled gNA are flowed over immobilized CasX protein. Alternatively, the ability to form an RNP can be assessed using in vitro cleavage assays against a defined target nucleic acid sequence.

j. gNA Stability

In some embodiments, a gNA variant has improved stability when compared to a reference gRNA. Increased stability and efficient folding may, in some embodiments, increase the extent to which a gNA variant persists inside a target cell, which may thereby increase the chance of forming a functional RNP capable of carrying out CasX functions such as gene editing. Increased stability of gNA variants may also, in some embodiments, allow for a similar outcome with a lower amount of gNA delivered to a cell, which may in turn reduce the chance of off-target effects during gene editing.

In other embodiments, the disclosure provides gNA in which the scaffold stem loop and/or the extended stem loop is replaced with a hairpin loop or a thermostable RNA stem loop in which the resulting gNA has increased stability and, depending on the choice of loop, can interact with certain cellular proteins or RNA. In some embodiments, the replacement RNA loop is selected from MS2, Qβ, U1 hairpin II, Uvsx, PP7, Phage replication loop, Kissing loop_a, Kissing loop_b1, Kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, Sarcin-ricin loop and Pseudoknots. Sequences of gNA variants including such components are provided in Table 2.

Guide NA stability can be assessed in a variety of ways, including for example in vitro by assembling the guide, incubating for varying periods of time in a solution that mimics the intracellular environment, and then measuring functional activity via the in vitro cleavage assays described herein. Alternatively, or in addition, gNAs can be harvested from cells at varying time points after initial transfection/transduction of the gNA to determine how long gNA variants persist relative to reference gRNAs.

k. Solubility

In some embodiments, a gNA variant has improved solubility when compared to a reference gRNA. In some embodiments, a gNA variant has improved solubility of the CasX protein:gNA RNP when compared to a reference gRNA. In some embodiments, solubility of the CasX protein:gNA RNP is improved by the addition of a ribozyme sequence to a 5′ or 3′ end of the gNA variant, for example the 5′ or 3′ of a reference sgRNA. Some ribozymes, such as the M1 ribozyme, can increase solubility of proteins through RNA mediated protein folding.

Increased solubility of CasX RNPs comprising a gNA variant as described herein can be evaluated through a variety of means known to one of skill in the art, such as by taking densitometry readings on a gel of the soluble fraction of lysed E. coli in which the CasX and gNA variants are expressed.

l. Resistance to Nuclease Activity

In some embodiments, a gNA variant has improved resistance to nuclease activity compared to a reference gRNA. Without wishing to be bound by any theory, increased resistance to nucleases, such as nucleases found in cells, may for example increase the persistence of a variant gNA in an intracellular environment, thereby improving gene editing.

Many nucleases are processive, and degrade RNA in a 3′ to 5′ fashion. Therefore, in some embodiments the addition of a nuclease resistant secondary structure to one or both termini of the gNA, or nucleotide changes that change the secondary structure of a sgNA, can produce gNA variants with increased resistance to nuclease activity. Resistance to nuclease activity may be evaluated through a variety of methods known to one of skill in the art. For example, in vitro methods of measuring resistance to nuclease activity may include for example contacting reference gNA and variants with one or more exemplary RNA nucleases and measuring degradation. Alternatively, or in addition, measuring persistence of a gNA variant in a cellular environment using the methods described herein can indicate the degree to which the gNA variant is nuclease resistant.

m. Binding Affinity to a Target DNA

In some embodiments, a gNA variant has improved affinity for the target DNA relative to a reference gRNA. In certain embodiments, a ribonucleoprotein complex comprising a gNA variant has improved affinity for the target DNA, relative to the affinity of an RNP comprising a reference gRNA. In some embodiments, the improved affinity of the RNP for the target DNA comprises improved affinity for the target sequence, improved affinity for the PAM sequence, improved ability of the RNP to search DNA for the target sequence, or any combinations thereof. In some embodiments, the improved affinity for the target DNA is the result of increased overall DNA binding affinity.

Without wishing to be bound by theory, it is possible that nucleotide changes in the gNA variant that affect the function of the OBD in the CasX protein may increase the affinity of CasX variant protein binding to the protospacer adjacent motif (PAM), as well as the ability to bind or utilize an increased spectrum of PAM sequences other than the canonical TTC PAM recognized by the reference CasX protein of SEQ ID NO: 2, including PAM sequences selected from the group consisting of TTC, ATC, GTC, and CTC, thereby increasing the affinity and diversity of the CasX variant protein for target DNA sequences, thereby increasing the target nucleic acid sequences that can be edited and/or bound, compared to a reference CasX. As described more fully, below, increasing the sequences of the target nucleic acid that can be edited, compared to a reference CasX, refers to both the PAM and the protospacer sequence and their directionality according to the orientation of the non-target strand. This does not imply that the PAM sequence of the non-target strand, rather than the target strand, is determinative of cleavage or mechanistically involved in target recognition. For example, when reference is to a TTC PAM, it may in fact be the complementary GAA sequence that is required for target cleavage, or it may be some combination of nucleotides from both strands. In the case of the CasX proteins disclosed herein, the PAM is located 5′ of the protospacer with at least a single nucleotide separating the PAM from the first nucleotide of the protospacer. Alternatively, or in addition, changes in the gNA that affect function of the helical I and/or helical II domains that increase the affinity of the CasX variant protein for the target DNA strand can increase the affinity of the CasX RNP comprising the variant gNA for target DNA.

n. Adding or Changing gNA Function

In some embodiments, gNA variants can comprise larger structural changes that change the topology of the gNA variant with respect to the reference gRNA, thereby allowing for different gNA functionality. For example, in some embodiments a gNA variant has swapped an endogenous stem loop of the reference gRNA scaffold with a previously identified stable RNA structure or a stem loop that can interact with a protein or RNA binding partner to recruit additional moieties to the CasX or to recruit CasX to a specific location, such as the inside of a viral capsid, that has the binding partner to the said RNA structure. In other scenarios the RNAs may be recruited to each other, as in Kissing loops, such that two CasX proteins can be co-localized for more effective gene editing at the target DNA sequence. Such RNA structures may include MS2, Qβ, U1 hairpin II, Uvsx, PP7, Phage replication loop, Kissing loop_a, Kissing loop_b1, Kissing loop_b2, G quadriplex M3q, G quadriplex telomere basket, Sarcin-ricin loop, or a Pseudoknot.

In some embodiments, a gNA variant comprises a terminal fusion partner. The term gNA variant is inclusive of variants that include exogenous sequences such as terminal fusions, or internal insertions. Exemplary terminal fusions may include fusion of the gRNA to a self-cleaving ribozyme or protein binding motif. As used herein, a “ribozyme” refers to an RNA or segment thereof with one or more catalytic activities similar to a protein enzyme. Exemplary ribozyme catalytic activities may include, for example, cleavage and/or ligation of RNA, cleavage and/or ligation of DNA, or peptide bond formation. In some embodiments, such fusions could either improve scaffold folding or recruit DNA repair machinery. For example, a gRNA may in some embodiments be fused to a hepatitis delta virus (HDV) antigenomic ribozyme, HDV genomic ribozyme, hatchet ribozyme (from metagenomic data), env25 pistol ribozyme (representative from Aliistipes putredinis), HH15 Minimal Hammerhead ribozyme, tobacco ringspot virus (TRSV) ribozyme, WT viral Hammerhead ribozyme (and rational variants), or Twisted Sister 1 or RBMX recruiting motif. Hammerhead ribozymes are RNA motifs that catalyze reversible cleavage and ligation reactions at a specific site within an RNA molecule. Hammerhead ribozymes include type I, type II and type III hammerhead ribozymes. The HDV, pistol, and hatchet ribozymes have self-cleaving activities. gNA variants comprising one or more ribozymes may allow for expanded gNA function as compared to a gRNA reference. For example, gNAs comprising self-cleaving ribozymes can, in some embodiments, be transcribed and processed into mature gNAs as part of polycistronic transcripts. Such fusions may occur at either the 5′ or the 3′ end of the gNA. In some embodiments, a gNA variant comprises a fusion at both the 5′ and the 3′ end, wherein each fusion is independently as described herein. In some embodiments, a gNA variant comprises a phage replication loop or a tetraloop. In some embodiments, a gNA comprises a hairpin loop that is capable of binding a protein. For example, in some embodiments the hairpin loop is an MS2, Qβ, U1 hairpin II, Uvsx, or PP7 hairpin loop.

In some embodiments, a gNA variant comprises one or more RNA aptamers. As used herein, an “RNA aptamer” refers to an RNA molecule that binds a target with high affinity and high specificity.

In some embodiments, a gNA variant comprises one or more riboswitches. As used herein, a “riboswitch” refers to an RNA molecule that changes state upon binding a small molecule.

In some embodiments, the gNA variant further comprises one or more protein binding motifs. Adding protein binding motifs to a reference gRNA or gNA variant of the disclosure may, in some embodiments, allow a CasX RNP to associate with additional proteins, which can for example add the functionality of those proteins to the CasX RNP.

IV. CasX Proteins for Modifying a Target Nucleic Acid

The term “CasX protein”, as used herein, refers to a family of proteins, and encompasses all naturally occurring CasX proteins, proteins that share at least 50% identity to naturally occurring CasX proteins, as well as CasX variants possessing one or more improved characteristics relative to a naturally-occurring reference CasX protein. Exemplary improved characteristics of the CasX variant embodiments include, but are not limited to improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target nucleic acid, improved ability to utilize a greater spectrum of PAM sequences in the editing and/or binding of target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased percentage of a eukaryotic genome that can be efficiently edited, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved protein:gNA (RNP) complex stability, improved protein solubility, improved protein:gNA (RNP) complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics, as described more fully, below. In the foregoing embodiments, the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 when assayed in a comparable fashion. In other embodiments, the improvement is at least about 1.1-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 when assayed in a comparable fashion.

The term CasX variant is inclusive of variants that are fusion proteins, i.e. the CasX is “fused to” a heterologous sequence. This includes CasX variants comprising CasX variant sequences and N-terminal, C-terminal, or internal fusions of the CasX to a heterologous protein or domain thereof.

CasX proteins of the disclosure comprise at least one of the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain, a helical II domain, an oligonucleotide binding domain (OBD), and a RuvC DNA cleavage domain (the last of which may be modified or deleted in a catalytically dead CasX variant), described more fully, below. Additionally, the CasX variant proteins of the disclosure have an enhanced ability to efficiently edit and/or bind target DNA utilizing PAM sequences selected from TTC, ATC, GTC, or CTC, compared to wild-type reference CasX proteins. In the foregoing, the PAM sequence is located at least 1 nucleotide 5′ to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX protein in a comparable assay system.

In some cases, the CasX protein is a naturally-occurring protein (e.g., naturally occurs in and is isolated from prokaryotic cells). In other embodiments, the CasX protein is not a naturally-occurring protein (e.g., the CasX protein is a CasX variant protein, a chimeric protein, and the like). A naturally-occurring CasX protein (referred to herein as a “reference CasX protein”) functions as an endonuclease that catalyzes a double strand break at a specific sequence in a targeted double-stranded DNA (dsDNA). The sequence specificity is provided by the targeting sequence of the associated gNA to which it is complexed, which hybridizes to a target sequence within the target nucleic acid.

In some embodiments, a CasX protein can bind and/or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and/or a polypeptide associated with target nucleic acid (e.g., methylation or acetylation of a histone tail). In some embodiments, the CasX protein is catalytically dead (dCasX) but retains the ability to bind a target nucleic acid. An exemplary catalytically dead CasX protein comprises one or more mutations in the active site of the RuvC domain of the CasX protein. In some embodiments, a catalytically dead CasX protein comprises substitutions at residues 672, 769 and/or 935 of SEQ ID NO: 1. In one embodiment, a catalytically dead CasX protein comprises substitutions of D672A, E769A and/or D935A in a reference CasX protein of SEQ ID NO: 1. In other embodiments, a catalytically dead CasX protein comprises substitutions at amino acids 659, 756 and/or 922 in a reference CasX protein of SEQ ID NO: 2. In some embodiments, a catalytically dead CasX protein comprises D659A, E756A and/or D922A substitutions in a reference CasX protein of SEQ ID NO: 2. In further embodiments, a catalytically dead CasX protein comprises deletions of all or part of the RuvC domain of the CasX protein. It will be understood that the same foregoing substitutions can similarly be introduced into the CasX variants of the disclosure, resulting in a dCasX variant. In one embodiment, all or a portion of the RuvC domain is deleted from the CasX variant, resulting in a dCasX variant. Catalytically inactive dCasX variant proteins can, in some embodiments, be used for base editing or epigenetic modifications. With a higher affinity for DNA, in some embodiments, catalytically inactive dCasX variant proteins can, relative to catalytically active CasX, find their target nucleic acid faster, remain bound to target nucleic acid for longer periods of time, bind target nucleic acid in a more stable fashion, or a combination thereof, thereby improving the function of the catalytically dead CasX variant protein.

a. Non-Target Strand Binding Domain

The reference CasX proteins of the disclosure comprise a non-target strand binding domain (NTSBD). The NTSBD is a domain not previously found in any Cas proteins; for example this domain is not present in Cas proteins such as Cas9, Cas12a/Cpf1, Cas13, Cas14, CASCADE, CSM, or CSY. Without being bound to theory or mechanism, a NTSBD in a CasX allows for binding to the non-target DNA strand and may aid in unwinding of the non-target and target strands. The NTSBD is presumed to be responsible for the unwinding, or the capture, of a non-target DNA strand in the unwound state. The NTSBD is in direct contact with the non-target strand in CryoEM model structures derived to date and may contain a non-canonical zinc finger domain. The NTSBD may also play a role in stabilizing DNA during unwinding, guide RNA invasion and R-loop formation. In some embodiments, an exemplary NTSBD comprises amino acids 101-191 of SEQ ID NO: 1 or amino acids 103-192 of SEQ ID NO: 2. In some embodiments, the NTSBD of a reference CasX protein comprises a four-stranded beta sheet.

b. Target Strand Loading Domain

The reference CasX proteins of the disclosure comprise a Target Strand Loading (TSL) domain. The TSL domain is a domain not found in certain Cas proteins such as Cas9, CASCADE, CSM, or CSY. Without wishing to be bound by theory or mechanism, it is thought that the TSL domain is responsible for aiding the loading of the target DNA strand into the RuvC active site of a CasX protein. In some embodiments, the TSL acts to place or capture the target-strand in a folded state that places the scissile phosphate of the target strand DNA backbone in the RuvC active site. The TSL comprises a cys4 (CXXC (SEQ ID NO: 246, CXXC (SEQ ID NO: 246) zinc finger/ribbon domain that is separated by the bulk of the TSL. In some embodiments, an exemplary TSL comprises amino acids 825-934 of SEQ ID NO: 1 or amino acids 813-921 of SEQ ID NO: 2.

c. Helical I Domain

The reference CasX proteins of the disclosure comprise a helical I domain. Certain Cas proteins other than CasX have domains that may be named in a similar way. However, in some embodiments, the helical I domain of a CasX protein comprises one or more unique structural features, or comprises a unique sequence, or a combination thereof, compared to non-CasX proteins. For example, in some embodiments, the helical I domain of a CasX protein comprises one or more unique secondary structures compared to domains in other Cas proteins that may have a similar name. For example, in some embodiments the helical I domain in a CasX protein comprises one or more alpha helices of unique structure and sequence in arrangement, number and length compared to other CRISPR proteins. In certain embodiments, the helical I domain is responsible for interacting with the bound DNA and spacer of the guide RNA. Without wishing to be bound by theory, it is thought that in some cases the helical I domain may contribute to binding of the protospacer adjacent motif (PAM). In some embodiments, an exemplary helical I domain comprises amino acids 57-100 and 192-332 of SEQ ID NO: 1, or amino acids 59-102 and 193-333 of SEQ ID NO: 2. In some embodiments, the helical I domain of a reference CasX protein comprises one or more alpha helices.

d. Helical II Domain

The reference CasX proteins of the disclosure comprise a helical II domain. Certain Cas proteins other than CasX have domains that may be named in a similar way. However, in some embodiments, the helical II domain of a CasX protein comprises one or more unique structural features, or a unique sequence, or a combination thereof, compared to domains in other Cas proteins that may have a similar name. For example, in some embodiments, the helical II domain comprises one or more unique structural alpha helical bundles that align along the target DNA:guide RNA channel. In some embodiments, in a CasX comprising a helical II domain, the target strand and guide RNA interact with helical II (and the helical I domain, in some embodiments) to allow RuvC domain access to the target DNA. The helical II domain is responsible for binding to the guide RNA scaffold stem loop as well as the bound DNA. In some embodiments, an exemplary helical II domain comprises amino acids 333-509 of SEQ ID NO: 1, or amino acids 334-501 of SEQ ID NO: 2.

e. Oligonucleotide Binding Domain

The reference CasX proteins of the disclosure comprise an Oligonucleotide Binding Domain (OBD). Certain Cas proteins other than CasX have domains that may be named in a similar way. However, in some embodiments, the OBD comprises one or more unique functional features, or comprises a sequence unique to a CasX protein, or a combination thereof. For example, in some embodiments the bridged helix (BH), helical I domain, helical II domain, and Oligonucleotide Binding Domain (OBD) together are responsible for binding of a CasX protein to the guide RNA. Thus, for example, in some embodiments the OBD is unique to a CasX protein in that it interacts functionally with a helical I domain, or a helical II domain, or both, each of which may be unique to a CasX protein as described herein. Specifically, in CasX the OBD largely binds the RNA triplex of the guide RNA scaffold. The OBD may also be responsible for binding to the protospacer adjacent motif (PAM). An exemplary OBD domain comprises amino acids 1-56 and 510-660 of SEQ ID NO: 1, or amino acids 1-58 and 502-647 of SEQ ID NO: 2.

f. RuvC DNA Cleavage Domain

The reference CasX proteins of the disclosure comprise a RuvC domain, that includes 2 partial RuvC domains (RuvC-I and RuvC-II). The RuvC domain is the ancestral domain of all type 12 CRISPR proteins. The RuvC domain originates from a TNPB (transposase B) like transposase. Similar to other RuvC domains, the CasX RuvC domain has a DED catalytic triad that is responsible for coordinating a magnesium (Mg) ion and cleaving DNA. In some embodiments, the RuvC has a DED motif active site that is responsible for cleaving both strands of DNA (one by one, most likely the non-target strand first at 11-14 nucleotides (nt) into the targeted sequence and then the target strand next at 2-4 nucleotides after the target sequence). Specifically in CasX, the RuvC domain is unique in that it is also responsible for binding the guide RNA scaffold stem loop that is critical for CasX function. An exemplary RuvC domain comprises amino acids 661-824 and 935-986 of SEQ ID NO: 1, or amino acids 648-812 and 922-978 of SEQ ID NO: 2.

g. Reference CasX Proteins

The disclosure provides reference CasX proteins. In some embodiments, a reference CasX protein is a naturally-occurring protein. For example, reference CasX proteins can be isolated from naturally occurring prokaryotes, such as Deltaproteobacteria, Planctomycetes, or Candidatus Sungbacteria species. A reference CasX protein (sometimes referred to herein as a reference CasX polypeptide) is a type II CRISPR/Cas endonuclease belonging to the CasX (sometimes referred to as Cas12e) family of proteins that is capable of interacting with a guide NA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex comprising the reference CasX protein can be targeted to a particular site in a target nucleic acid via base pairing between the targeting sequence (or spacer) of the gNA and a target sequence in the target nucleic acid. In some embodiments, the RNP comprising the reference CasX protein is capable of cleaving target DNA. In some embodiments, the RNP comprising the reference CasX protein is capable of nicking target DNA. In some embodiments, the RNP comprising the reference CasX protein is capable of editing target DNA, for example in those embodiments where the reference CasX protein is capable of cleaving or nicking DNA, followed by non-homologous end joining (NHEJ), homology-directed repair (HDR), homology-independent targeted integration (HITI), micro-homology mediated end joining (MMEJ), single strand annealing (SSA) or base excision repair (BER). In some embodiments, the RNP comprising the CasX protein is a catalytically dead (is catalytically inactive or has substantially no cleavage activity) CasX protein (dCasX), but retains the ability to bind the target DNA, described more fully, supra.

In some cases, a reference CasX protein is isolated or derived from Deltaproteobacteria. In some embodiments, a CasX protein comprises a sequence at least 50% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical or 100% identical to a sequence of:

(SEQ ID NO: 1)   1 MEKRINKIRK KLSADNATKP VSRSGPMKTL LVRVMTDDLK KRLEKRRKKP EVMPQVISNN  61 AANNLRMLLD DYTKMKEAIL QVYWQEFKDD HVGLMCKFAQ PASKKIDQNK LKPEMDEKGN 121 LTTAGFACSQ CGQPLFVYKL EQVSEKGKAY TNYFGRCNVA EHEKLILLAQ LKPEKDSDEA 181 VTYSLGKFGQ RALDFYSIHV TKESTHPVKP LAQIAGNRYA SGPVGKALSD ACMGTIASFL 241 SKYQDIIIEH QKVVKGNQKR LESLRELAGK ENLEYPSVTL PPQPHTKEGV DAYNEVIARV 301 RMWVNLNLWQ KLKLSRDDAK PLLRLKGFPS FPVVERRENE VDWWNTINEV KKLIDAKRDM 361 GRVFWSGVTA EKRNTILEGY NYLPNENDHK KREGSLENPK KPAKRQFGDL LLYLEKKYAG 421 DWGKVFDEAW ERIDKKIAGL TSHIEREEAR NAEDAQSKAV LTDWLRAKAS FVLERLKEMD 481 EKEFYACEIQ LQKWYGDLRG NPFAVEAENR VVDISGFSIG SDGHSIQYRN LLAWKYLENG 541 KREFYLLMNY GKKGRIRFTD GTDIKKSGKW QGLLYGGGKA KVIDLTFDPD DEQLIILPLA 601 FGTRQGREFI WNDLLSLETG LIKLANGRVI EKTIYNKKIG RDEPALFVAL TFERREVVDP 661 SNIKPVNLIG VDRGENIPAV IALTDPEGCP LPEFKDSSGG PTDILRIGEG YKEKQRAIQA 721 AKEVEQRRAG GYSRKFASKS RNLADDMVRN SARDLFYHAV THDAVLVFEN LSRGFGRQGK 781 RTFMTERQYT KMEDWLTAKL AYEGLTSKTY LSKTLAQYTS KTCSNCGFTI TTADYDGMLV 841 RLKKTSDGWA TTLNNKELKA EGQITYYNRY KRQTVEKELS AELDRLSEES GNNDISKWTK 901 GRRDEALFLL KKRFSHRPVQ EQFVCLDCGH EVHADEQAAL NIARSWLFLN SNSTEFKSYK 961 SGKQPFVGAW QAFYKRRLKE VWKPNA.

In some cases, a reference CasX protein is isolated or derived from Planctomycetes. In some embodiments, a CasX protein comprises a sequence at least 50% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical or 100% identical to a sequence of:

(SEQ ID NO: 2) 1 MQEIKRINKI RRRLVKDSNT KKAGKTGPMK TLLVRVMTPD LRERLENLRK KPENIPQPIS  61 NTSRANLNKL LTDYTEMKKA ILHVYWEEFO KDPVGLMSRV AOPAPHNIDO RKLIPVKDGN  121 ERLTSSGFAC SQCCQPLYVY KLEQVNDKGK PHTNYFGRCN VSEHEPLILL SPHKPEANDE  181 LVTYSLGKFG QRALDFYSIH VTPESNHPVK PLEQIGGNSC ASGPVGKALS DACMGAVASF  241 LTKYODIILE HQKVIKKNEK RLANLKDIAS ANGLAFPKIT LPPQPHTKEG IEAYNNVVAQ  301 IVIWVNLNLW QKLKIGRDEA KPLQRLKGFP SFPLVERQAN EVDWWDMVCN VKKLINEKKE  361 DGKVFWQNLA GYKRQEALLP YLSSEEDRKK GNKFARYQFG DLLLHLEKKH GEDWGKVYDE  421 AWERIDKKVE GLSKHIKLEE ERRSEDAQSK AALTDWLPAK ASFVIEGLKE ADKDEFCRCE  481 LKLQKWYGDL PGKPFAIEAE NSILDISGFS KQYNCAFIWQ KDGVKKLNLY LIINYFKGGK  541 LRFKKIKPEA FEANRFYTVI NKKSGEIVPM EVNFNFDDPN LIILPLAFGK RQGREFIWND  601 LLSLETGSLK LANGRVIEKT LYNRRTRQDE RALFVALTFE RREVLDSSNI KPMNLIGIDR  661 GENIPAVIAL TDPEGCPLSR FKDSLGNPTH ILRIGESYKE KQRTIQAAKE VEQRPAGGYS 721 RKYASKAKNL ADEMVRNTAR DLLYYAVTQD AMLIFENLSR GEGRQGKRTF MAERQYTRME  781 DWLTAKLAYE GLPSKTYLSK TLATZTSKTC SNCGFTITSA DYDRVLEKLK KTATGWMTTI  841 NGKELKVEGQ ITYYNPYKRQ NVVKDLSVEL DRLSEESVNN DISSWTKGRS GEALSLLKKR  901 FSHRPVQEKF VCLNCGFETH ADEQAALNIA RSWLFIRSQE YKKYQTNKTT GNTDKRAFVE  961 TWQSFYRKKL KEVWKPAV. 

In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 2, or at least 60% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 2, or at least 80% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 2, or at least 90% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 2, or at least 95% similarity thereto. In some embodiments, the CasX protein consists of the sequence of SEQ ID NO: 2. In some embodiments, the CasX protein comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40 or at least 50 mutations relative to the sequence of SEQ ID NO: 2. These mutations can be insertions, deletions, amino acid substitutions, or any combinations thereof.

In some cases, a reference CasX protein is isolated or derived from Candidatus Sungbacteria. In some embodiments, a CasX protein comprises a sequence at least 50% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical or 100% identical to a sequence of

(SEQ ID NO: 3)  1 MDNANKPSTK SLVNTTRISD HFGVTPGQVT RVFSFGIIPT KRQYAIIERW FAAVEAARER  61 LYGMIYARFQ ENPPAYIKEK FSYETFFKGR PVLNGLRDID PTIMTSAVFT ALRHKAEGAM  121 AAFHTNERRL FEEARKKMRE YAECLKANEA LURGAAD1DW DKIVNALRTR LNTCLAPEYD  181 AVIADFGALC AFRALIAETN ALKGAYNHAL NQMLPALVKV DEPEEAEESP RLRFFNGRIN  241 DLPHFPVAER ETPPDTETII RQLEDMARVI PDTAEILGYI HRIRHKAARR KPGSAVPLPQ  301 RVALYCAIRM ERNPEEDPST VAGHFLGEID RVCEKPRQGL VPTPFDSQIR ARYMDIISFR  361 ATLAHPDRWT EIQFLRSNAA SRRVRAETIS APFEGFSWTS NRTNPAPQYG MALAKDANAP  421 ADAPELCICL SPSSAAFSVR EKGGDLIYMR PTGGRRGKDN PGKEITWVPG SFDEYPASGV 481 ALKLRLYFGR SQARRMLTNN TWGLLSDNPR VFAANAELVG KKRNPQDRWK LFFHMVISGP  541 PPVEYLDFSS DVRSRARTVI GINRGEVNPL AYAVVSVEDG QVLEEGLLGK KEYIDQLIET  601 RRPISEYQSR EQTPPRDLRQ RVPHLQDTVL GSARAKIHSL IAFWKGILAI ERLDDQFHGR  661 EQKIIPKKTY LANKTGFMNA LSFSGAVRVD KEGNPWGGMI ETYPGGISRT CTQCGTVWLA  721 RRPKNPGHRD ANVVIPDIVD DAAATGFDNV DCDAGTVDYG ELFTLSREWV RLTPPYSRVM  781 RGTLGDLEPA IRQGDDRKSR QMLELALEPQ PQWGQFECHR CGFNGQSDVL AATNLARRAI  841 SLIRRLPDTD TPPTP. 

In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 3, or at least 60% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 3, or at least 80% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 3, or at least 90% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 3, or at least 95% similarity thereto. In some embodiments, the CasX protein consists of the sequence of SEQ ID NO: 3. In some embodiments, the CasX protein comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40 or at least 50 mutations relative to the sequence of SEQ ID NO: 3. These mutations can be insertions, deletions, amino acid substitutions, or any combinations thereof.

h. CasX Variant Proteins

The present disclosure provides variants of a reference CasX protein (interchangeably referred to herein as “CasX variant” or “CasX variant protein”), wherein the CasX variants comprise at least one modification in at least one domain relative to the reference CasX protein, including but not limited to the sequences of SEQ ID NOS:1-3. In some embodiments, the CasX variant exhibits at least one improved characteristic compared to the reference CasX protein. All variants that improve one or more functions or characteristics of the CasX variant protein when compared to a reference CasX protein described herein are envisaged as being within the scope of the disclosure. In some embodiments, the modification is a mutation in one or more amino acids of the reference CasX. In other embodiments, the modification is a substitution of one or more domains of the reference CasX with one or more domains from a different CasX. In some embodiments, insertion includes the insertion of a part or all of a domain from a different CasX protein. Mutations can occur in any one or more domains of the reference CasX protein, and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain of the reference CasX protein. The domains of CasX proteins include the non-target strand binding (NTSB) domain, the target strand loading (TSL) domain, the helical I domain, the helical II domain, the oligonucleotide binding domain (OBD), and the RuvC DNA cleavage domain. Any change in amino acid sequence of a reference CasX protein that leads to an improved characteristic of the CasX protein is considered a CasX variant protein of the disclosure. For example, CasX variants can comprise one or more amino acid substitutions, insertions, deletions, or swapped domains, or any combinations thereof, relative to a reference CasX protein sequence.

In some embodiments, the CasX variant protein comprises at least one modification in at least each of two domains of the reference CasX protein, including the sequences of SEQ ID NOS: 1-3. In some embodiments, the CasX variant protein comprises at least one modification in at least 2 domains, in at least 3 domains, at least 4 domains or at least 5 domains of the reference CasX protein. In some embodiments, the CasX variant protein comprises two or more modifications in at least one domain of the reference CasX protein. In some embodiments, the CasX variant protein comprises at least two modifications in at least one domain of the reference CasX protein, at least three modifications in at least one domain of the reference CasX protein or at least four modifications in at least one domain of the reference CasX protein. In some embodiments, wherein the CasX variant comprises two or more modifications compared to a reference CasX protein, each modification is made in a domain independently selected from the group consisting of a NTSBD, TSLD, helical I domain, helical II domain, OBD, and RuvC DNA cleavage domain.

In some embodiments, the at least one modification of the CasX variant protein comprises a deletion of at least a portion of one domain of the reference CasX protein. In some embodiments, the deletion is in the NTSBD, TSLD, helical I domain, helical II domain, OBD, or RuvC DNA cleavage domain.

Suitable mutagenesis methods for generating CasX variant proteins of the disclosure may include, for example, Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping. Exemplary methods for the generation of CasX variants with improved characteristics are provided in the Examples, below. In some embodiments, the CasX variants are designed, for example by selecting one or more desired mutations in a reference CasX. In certain embodiments, the activity of a reference CasX protein is used as a benchmark against which the activity of one or more CasX variants are compared, thereby measuring improvements in function of the CasX variants. Exemplary improvements of CasX variants include, but are not limited to, improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target DNA, improved ability to utilize a greater spectrum of PAM sequences in the editing or binding of target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved CasX:gNA (RNP) complex stability, improved protein solubility, improved CasX:gNA (RNP) complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics, as described more fully, below.

In some embodiments of the CasX variants described herein, the at least one modification comprises: (a) a substitution of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant; (b) a deletion of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant; (c) an insertion of 1 to 100 consecutive or non-consecutive amino acids in the CasX; or (d) any combination of (a)-(c). In some embodiments, the at least one modification comprises: (a) a substitution of 5-10 consecutive or non-consecutive amino acids in the CasX variant. (b) a deletion of 1-5 consecutive or non-consecutive amino acids in the CasX variant; (c) an insertion of 1-5 consecutive or non-consecutive amino acids in the CasX; or (d) any combination of (a)-(c).

In some embodiments, the CasX variant protein comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40 or at least 50 mutations relative to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. These mutations can be insertions, deletions, amino acid substitutions, or any combinations thereof.

In some embodiments, the CasX variant protein comprises at least one amino acid substitution in at least one domain of a reference CasX protein. In some embodiments, the CasX variant protein comprises at least about 1-4 amino acid substitutions, 1-10 amino acid substitutions, 1-20 amino acid substitutions, 1-30 amino acid substitutions, 1-40 amino acid substitutions, 1-50 amino acid substitutions, 1-60 amino acid substitutions, 1-70 amino acid substitutions, 1-80 amino acid substitutions, 1-90 amino acid substitutions, 1-100 amino acid substitutions, 2-10 amino acid substitutions, 2-20 amino acid substitutions, 2-30 amino acid substitutions, 3-10 amino acid substitutions, 3-20 amino acid substitutions, 3-30 amino acid substitutions, 4-10 amino acid substitutions, 4-20 amino acid substitutions, 3-300 amino acid substitutions, 5-10 amino acid substitutions, 5-20 amino acid substitutions, 5-30 amino acid substitutions, 10-50 amino acid substitutions, or 20-50 amino acid substitutions, relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises at least about 100 amino acid substitutions relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in a single domain relative to the reference CasX protein. In some embodiments, the amino acid substitutions are conservative substitutions. In other embodiments, the substitutions are non-conservative; e.g., a polar amino acid is substituted for a non-polar amino acid, or vice versa.

In some embodiments, a CasX variant protein comprises 1 amino acid substitution, 2-3 consecutive amino acid substitutions, 2-4 consecutive amino acid substitutions, 2-5 consecutive amino acid substitutions, 2-6 consecutive amino acid substitutions, 2-7 consecutive amino acid substitutions, 2-8 consecutive amino acid substitutions, 2-9 consecutive amino acid substitutions, 2-10 consecutive amino acid substitutions, 2-20 consecutive amino acid substitutions, 2-30 consecutive amino acid substitutions, 2-40 consecutive amino acid substitutions, 2-50 consecutive amino acid substitutions, 2-60 consecutive amino acid substitutions, 2-70 consecutive amino acid substitutions, 2-80 consecutive amino acid substitutions, 2-90 consecutive amino acid substitutions, 2-100 consecutive amino acid substitutions, 3-10 consecutive amino acid substitutions, 3-20 consecutive amino acid substitutions, 3-30 consecutive amino acid substitutions, 4-10 consecutive amino acid substitutions, 4-20 consecutive amino acid substitutions, 3-300 consecutive amino acid substitutions, 5-10 consecutive amino acid substitutions, 5-20 consecutive amino acid substitutions, 5-30 consecutive amino acid substitutions, 10-50 consecutive amino acid substitutions or 20-50 consecutive amino acid substitutions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acid substitutions. In some embodiments, a CasX variant protein comprises a substitution of at least about 100 consecutive amino acids. As used herein “consecutive amino acids” refer to amino acids that are contiguous in the primary sequence of a polypeptide.

In some embodiments, a CasX variant protein comprises two or more substitutions relative to a reference CasX protein, and the two or more substitutions are not in consecutive amino acids of the reference CasX sequence. For example, a first substitution may be in a first domain of the reference CasX protein, and a second substitution may be in a second domain of the reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 non-consecutive substitutions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises at least 20 non-consecutive substitutions relative to a reference CasX protein. Each non-consecutive substitution may be of any length of amino acids described herein, e.g., 1-4 amino acids, 1-10 amino acids, and the like. In some embodiments, the two or more substitutions relative to the reference CasX protein are not the same length, for example, one substitution is one amino acid and a second substitution is three amino acids. In some embodiments, the two or more substitutions relative to the reference CasX protein are the same length, for example both substitutions are two consecutive amino acids in length.

Any amino acid can be substituted for any other amino acid in the substitutions described herein. The substitution can be a conservative substitution (e.g., a basic amino acid is substituted for another basic amino acid). The substitution can be a non-conservative substitution (e.g., a basic amino acid is substituted for an acidic amino acid or vice versa). For example, a proline in a reference CasX protein can be substituted for any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine or valine to generate a CasX variant protein of the disclosure.

In some embodiments, a CasX variant protein comprises at least one amino acid deletion relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises a deletion of 1-4 amino acids, 1-10 amino acids, 1-20 amino acids, 1-30 amino acids, 1-40 amino acids, 1-50 amino acids, 1-60 amino acids, 1-70 amino acids, 1-80 amino acids, 1-90 amino acids, 1-100 amino acids, 2-10 amino acids, 2-20 amino acids, 2-30 amino acids, 3-10 amino acids, 3-20 amino acids, 3-30 amino acids, 4-10 amino acids, 4-20 amino acids, 3-300 amino acids, 5-10 amino acids, 5-20 amino acids, 5-30 amino acids, 10-50 amino acids or 20-50 amino acids relative to a reference CasX protein. In some embodiments, a CasX variant comprises a deletion of at least about 100 consecutive amino acids relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or 100 consecutive amino acids relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids.

In some embodiments, a CasX variant protein comprises two or more deletions relative to a reference CasX protein, and the two or more deletions are not consecutive amino acids. For example, a first deletion may be in a first domain of the reference CasX protein, and a second deletion may be in a second domain of the reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 non-consecutive deletions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises at least 20 non-consecutive deletions relative to a reference CasX protein. Each non-consecutive deletion may be of any length of amino acids described herein, e.g., 1-4 amino acids, 1-10 amino acids, and the like.

In some embodiments, the CasX variant protein comprises at least one amino acid insertion. In some embodiments, a CasX variant protein comprises an insertion of 1 amino acid, an insertion of 2-3 consecutive amino acids, 2-4 consecutive amino acids, 2-5 consecutive amino acids, 2-6 consecutive amino acids, 2-7 consecutive amino acids, 2-8 consecutive amino acids, 2-9 consecutive amino acids, 2-10 consecutive amino acids, 2-20 consecutive amino acids, 2-30 consecutive amino acids, 2-40 consecutive amino acids, 2-50 consecutive amino acids, 2-60 consecutive amino acids, 2-70 consecutive amino acids, 2-80 consecutive amino acids, 2-90 consecutive amino acids, 2-100 consecutive amino acids, 3-10 consecutive amino acids, 3-20 consecutive amino acids, 3-30 consecutive amino acids, 4-10 consecutive amino acids, 4-20 consecutive amino acids, 3-300 consecutive amino acids, 5-10 consecutive amino acids, 5-20 consecutive amino acids, 5-30 consecutive amino acids, 10-50 consecutive amino acids or 20-50 consecutive amino acids relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises an insertion of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids. In some embodiments, a CasX variant protein comprises an insertion of at least about 100 consecutive amino acids.

In some embodiments, a CasX variant protein comprises two or more insertions relative to a reference CasX protein, and the two or more insertions are not consecutive amino acids of the sequence. For example, a first insertion may be in a first domain of the reference CasX protein, and a second insertion may be in a second domain of the reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 non-consecutive insertions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises at least 10 to about 20 or more non-consecutive insertions relative to a reference CasX protein. Each non-consecutive insertion may be of any length of amino acids described herein, e.g., 1-4 amino acids, 1-10 amino acids, and the like.

Any amino acid, or combination of amino acids, can be inserted as described herein. For example, a proline, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine or valine or any combination thereof can be inserted into a reference CasX protein of the disclosure to generate a CasX variant protein.

Any permutation of the substitution, insertion and deletion embodiments described herein can be combined to generate a CasX variant protein of the disclosure. For example, a CasX variant protein can comprise at least one substitution and at least one deletion relative to a reference CasX protein sequence, at least one substitution and at least one insertion relative to a reference CasX protein sequence, at least one insertion and at least one deletion relative to a reference CasX protein sequence, or at least one substitution, one insertion and one deletion relative to a reference CasX protein sequence.

In some embodiments, the CasX variant protein has at least about 60% sequence similarity, at least 70% similarity, at least 80% similarity, at least 85% similarity, at least 86% similarity, at least 87% similarity, at least 88% similarity, at least 89% similarity, at least 90% similarity, at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, at least 99% similarity, at least 99.5% similarity, at least 99.6% similarity, at least 99.7% similarity, at least 99.8% similarity or at least 99.9% similarity to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, the CasX variant protein has at least about 60% sequence similarity to SEQ ID NO: 2 or a portion thereof. In some embodiments, the CasX variant protein comprises a substitution of Y789T of SEQ ID NO: 2, a deletion of P793 of SEQ ID NO: 2, a substitution of Y789D of SEQ ID NO: 2, a substitution of T72S of SEQ ID NO: 2, a substitution of I546V of SEQ ID NO: 2, a substitution of E552A of SEQ ID NO: 2, a substitution of A636D of SEQ ID NO: 2, a substitution of F536S of SEQ ID NO:2, a substitution of A708K of SEQ ID NO: 2, a substitution of Y797L of SEQ ID NO: 2, a substitution of L792G SEQ ID NO: 2, a substitution of A739V of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO: 2, an insertion of A at position 661of SEQ ID NO: 2, a substitution of A788W of SEQ ID NO: 2, a substitution of K390R of SEQ ID NO: 2, a substitution of A751S of SEQ ID NO: 2, a substitution of E385A of SEQ ID NO: 2, an insertion of P at position 696 of SEQ ID NO: 2, an insertion of M at position 773 of SEQ ID NO: 2, a substitution of G695H of SEQ ID NO: 2, an insertion of AS at position 793 of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, a substitution of C477R of SEQ ID NO: 2, a substitution of C477K of SEQ ID NO: 2, a substitution of C479A of SEQ ID NO: 2, a substitution of C479L of SEQ ID NO: 2, a substitution of I55F of SEQ ID NO: 2, a substitution of K210R of SEQ ID NO: 2, a substitution of C233S of SEQ ID NO: 2, a substitution of D231N of SEQ ID NO: 2, a substitution of Q338E of SEQ ID NO: 2, a substitution of Q338R of SEQ ID NO: 2, a substitution of L379R of SEQ ID NO: 2, a substitution of K390R of SEQ ID NO: 2, a substitution of L481Q of SEQ ID NO: 2, a substitution of F495S of SEQ ID NO:2, a substitution of D600N of SEQ ID NO: 2, a substitution of T886K of SEQ ID NO: 2, a substitution of A739V of SEQ ID NO: 2, a substitution of K460N of SEQ ID NO: 2, a substitution of I199F of SEQ ID NO: 2, a substitution of G492P of SEQ ID NO: 2, a substitution of T153I of SEQ ID NO: 2, a substitution of R591I of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, an insertion of AS at position 796 of SEQ ID NO:2, an insertion of L at position 889 of SEQ ID NO: 2, a substitution of E121D of SEQ ID NO: 2, a substitution of S270W of SEQ ID NO: 2, a substitution of E712Q of SEQ ID NO: 2, a substitution of K942Q of SEQ ID NO: 2, a substitution of E552K of SEQ ID NO:2, a substitution of K25Q of SEQ ID NO: 2, a substitution of N47D of SEQ ID NO: 2, an insertion of T at position 6% of SEQ ID NO: 2, a substitution of L685I of SEQ ID NO: 2, a substitution of N880D of SEQ ID NO: 2, a substitution of Q102R of SEQ ID NO: 2, a substitution of M734K of SEQ ID NO: 2, a substitution of A724S of SEQ ID NO: 2, a substitution of T704K of SEQ ID NO: 2, a substitution of P224K of SEQ ID NO: 2, a substitution of K25R of SEQ ID NO: 2, a substitution of M29E of SEQ ID NO: 2, a substitution of H152D of SEQ ID NO: 2, a substitution of S219R of SEQ ID NO: 2, a substitution of E475K of SEQ ID NO: 2, a substitution of G226R of SEQ ID NO: 2, a substitution of A377K of SEQ ID NO: 2, a substitution of E480K of SEQ ID NO: 2, a substitution of K416E of SEQ ID NO: 2, a substitution of H164R of SEQ ID NO: 2, a substitution of K767R of SEQ ID NO: 2, a substitution of I7F of SEQ ID NO: 2, a substitution of M29R of SEQ ID NO: 2, a substitution of H435R of SEQ ID NO: 2, a substitution of E385Q of SEQ ID NO: 2, a substitution of E385K of SEQ ID NO: 2, a substitution of I279F of SEQ ID NO: 2, a substitution of D489S of SEQ ID NO: 2, a substitution of D732N of SEQ ID NO: 2, a substitution of A739T of SEQ ID NO: 2, a substitution of W885R of SEQ ID NO: 2, a substitution of E53K of SEQ ID NO: 2, a substitution of A238T of SEQ ID NO: 2, a substitution of P283Q of SEQ ID NO: 2, a substitution of E292K of SEQ ID NO: 2, a substitution of Q628E of SEQ ID NO: 2, a substitution of R388Q of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO: 2, a substitution of L792K of SEQ ID NO: 2, a substitution of L792E of SEQ ID NO: 2, a substitution of M779N of SEQ ID NO: 2, a substitution of G27D of SEQ ID NO: 2, a substitution of K955R of SEQ ID NO: 2, a substitution of S867R of SEQ ID NO: 2, a substitution of R693I of SEQ ID NO: 2, a substitution of F189Y of SEQ ID NO: 2, a substitution of V635M of SEQ ID NO: 2, a substitution of F399L of SEQ ID NO: 2, a substitution of E498K of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO: 2, a substitution of V254G of SEQ ID NO: 2, a substitution of P793S of SEQ ID NO: 2, a substitution of K188E of SEQ ID NO: 2, a substitution of QT945KI of SEQ ID NO: 2, a substitution of T620P of SEQ ID NO: 2, a substitution of T946P of SEQ ID NO: 2, a substitution of TI949PP of SEQ ID NO: 2, a substitution of N952T of SEQ ID NO: 2, a substitution of K682E of SEQ ID NO: 2, a substitution of K975R of SEQ ID NO: 2, a substitution of L212P of SEQ ID NO: 2, a substitution of E292R of SEQ ID NO: 2, a substitution of I303K of SEQ ID NO: 2, a substitution of C349E of SEQ ID NO: 2, a substitution of E385P of SEQ ID NO: 2, a substitution of E386N of SEQ ID NO: 2, a substitution of D387K of SEQ ID NO: 2, a substitution of L404K of SEQ ID NO: 2, a substitution of E466H of SEQ ID NO: 2, a substitution of C477Q of SEQ ID NO: 2, a substitution of C477H of SEQ ID NO: 2, a substitution of C479A of SEQ ID NO: 2, a substitution of D659H of SEQ ID NO: 2, a substitution of T806V of SEQ ID NO: 2, a substitution of K808S of SEQ ID NO: 2, an insertion of AS at position 797 of SEQ ID NO: 2, a substitution of V959M of SEQ ID NO: 2, a substitution of K975Q of SEQ ID NO: 2, a substitution of W974G of SEQ ID NO: 2, a substitution of A708Q of SEQ ID NO: 2, a substitution of V711K of SEQ ID NO: 2, a substitution of D733T of SEQ ID NO: 2, a substitution of L742W of SEQ ID NO: 2, a substitution of V747K of SEQ ID NO: 2, a substitution of F755M of SEQ ID NO: 2, a substitution of M771A of SEQ ID NO: 2, a substitution of M771Q of SEQ ID NO: 2, a substitution of W782Q of SEQ ID NO: 2, a substitution of G791F, of SEQ ID NO: 2, a substitution of L792D of SEQ ID NO: 2, a substitution of L792K of SEQ ID NO: 2, a substitution of P793Q of SEQ ID NO: 2, a substitution of P793G of SEQ ID NO: 2, a substitution of Q804A of SEQ ID NO: 2, a substitution of Y966N of SEQ ID NO: 2, a substitution of Y723N of SEQ ID NO: 2, a substitution of Y857R of SEQ ID NO: 2, a substitution of S890R of SEQ ID NO: 2, a substitution of S932M of SEQ ID NO: 2, a substitution of L897M of SEQ ID NO: 2, a substitution of R624G of SEQ ID NO: 2, a substitution of S603G of SEQ ID NO: 2, a substitution of N737S of SEQ ID NO: 2, a substitution of L307K of SEQ ID NO: 2, a substitution of I658V of SEQ ID NO: 2, an insertion of PT at position 688 of SEQ ID NO: 2, an insertion of SA at position 794 of SEQ ID NO: 2, a substitution of S877R of SEQ ID NO: 2, a substitution of N580T of SEQ ID NO: 2, a substitution of V335G of SEQ ID NO: 2, a substitution of T620S of SEQ ID NO: 2, a substitution of W345G of SEQ ID NO: 2, a substitution of T280S of SEQ ID NO: 2, a substitution of L406P of SEQ ID NO: 2, a substitution of A612D of SEQ ID NO: 2, a substitution of A751S of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO: 2, a substitution of V351M of SEQ ID NO: 2, a substitution of K210N of SEQ ID NO: 2, a substitution of D40A of SEQ ID NO: 2, a substitution of E773G of SEQ ID NO: 2, a substitution of H207L of SEQ ID NO: 2, a substitution of T62A SEQ ID NO: 2, a substitution of T287P of SEQ ID NO: 2, a substitution of T832A of SEQ ID NO: 2, a substitution of A893S of SEQ ID NO: 2, an insertion of V at position 14 of SEQ ID NO: 2, an insertion of AG at position 13 of SEQ ID NO: 2, a substitution of R11V of SEQ ID NO: 2, a substitution of R12N of SEQ ID NO: 2, a substitution of R13H of SEQ ID NO: 2, an insertion of Y at position 13 of SEQ ID NO: 2, a substitution of R12L of SEQ ID NO: 2, an insertion of Q at position 13 of SEQ ID NO: 2, an substitution of V15S of SEQ ID NO: 2, an insertion of D at position 17 of SEQ ID NO: 2 or a combination thereof.

In some embodiments, the CasX variant comprises at least one modification in the NTSB domain.

In some embodiments, the CasX variant comprises at least one modification in the TSL domain. In some embodiments, the at least one modification in the TSL domain comprises an amino acid substitution of one or more of amino acids Y857, S890, or S932 of SEQ ID NO: 2.

In some embodiments, the CasX variant comprises at least one modification in the helical I domain. In some embodiments, the at least one modification in the helical I domain comprises an amino acid substitution of one or more of amino acids S219, L249, E259, Q252, E292, L307, or D318 of SEQ ID NO: 2.

In some embodiments, the CasX variant comprises at least one modification in the helical II domain. In some embodiments, the at least one modification in the helical II domain comprises an amino acid substitution of one or more of amino acids D361, L379, E385, E386, D387, F399, L404, R458, C477, or D489 of SEQ ID NO: 2.

In some embodiments, the CasX variant comprises at least one modification in the OBD domain. In some embodiments, the at least one modification in the OBD comprises an amino acid substitution of one or more of amino acids F536, E552, T620, or I658 of SEQ ID NO: 2.

In some embodiments, the CasX variant comprises at least one modification in the RuvC DNA cleavage domain. In some embodiments, the at least one modification in the RuvC DNA cleavage domain comprises an amino acid substitution of one or more of amino acids K682, G695, A708, V711, D732, A739, D733, L742, V747, F755, M771, M779, W782, A788, G791, L792, P793, Y797, M799, Q804, S819, or Y857 or a deletion of amino acid P793 of SEQ ID NO: 2.

In some embodiments, the CasX variant comprises at least one modification compared to the reference CasX sequence of SEQ ID NO: 2 is selected from one or more of: (a) an amino acid substitution of L379R; (b) an amino acid substitution of A708K; (c) an amino acid substitution of T620P; (d) an amino acid substitution of E385P; (e) an amino acid substitution of Y857R; (f) an amino acid substitution of I658V; (g) an amino acid substitution of F399L; (h) an amino acid substitution of Q252K; (i) an amino acid substitution of L404K; and (j) an amino acid deletion of P793.

In some embodiments, a CasX variant protein comprises at least two amino acid changes to a reference CasX protein amino acid sequence. The at least two amino acid changes can be substitutions, insertions, or deletions of a reference CasX protein amino acid sequence, or any combination thereof. The substitutions, insertions or deletions can be any substitution, insertion or deletion in the sequence of a reference CasX protein described herein. In some embodiments, the changes are contiguous, non-contiguous, or a combination of contiguous and non-contiguous amino acid changes to a reference CasX protein sequence. In some embodiments, the reference CasX protein is SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 or at least 100 amino acid changes to a reference CasX protein sequence. In some embodiments, a CasX variant protein comprises 1-50, 3-40, 5-30, 5-20, 5-15, 5-10, 10-50, 10-40, 10-30, 10-20, 15-50, 15-40, 15-30, 2-25, 2-24, 2-22, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-25, 3-24, 3-22, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-25, 4-24, 4-22, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-25, 5-24, 5-22, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7 or 5-6 amino acid changes to a reference CasX protein sequence. In some embodiments, a CasX variant protein comprises 15-20 changes to a reference CasX protein sequence. In some embodiments, a CasX variant protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid changes to a reference CasX protein sequence. In some embodiments, the at least two amino acid changes to the sequence of a reference CasX variant protein are selected from the group consisting of: a substitution of Y789T of SEQ ID NO: 2, a deletion of P793 of SEQ ID NO: 2, a substitution of Y789D of SEQ ID NO: 2, a substitution of T72S of SEQ ID NO: 2, a substitution of I546V of SEQ ID NO: 2, a substitution of E552A of SEQ ID NO: 2, a substitution of A636D of SEQ ID NO: 2, a substitution of F536S of SEQ ID NO:2, a substitution of A708K of SEQ ID NO: 2, a substitution of Y797L of SEQ ID NO: 2, a substitution of L792G SEQ ID NO: 2, a substitution of A739V of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO: 2, an insertion of A at position 661 of SEQ ID NO: 2, a substitution of A788W of SEQ ID NO: 2, a substitution of K390R of SEQ ID NO: 2, a substitution of A751S of SEQ ID NO: 2, a substitution of E385A of SEQ ID NO: 2, an insertion of P at position 696 of SEQ ID NO: 2, an insertion of M at position 773 of SEQ ID NO: 2, a substitution of G695H of SEQ ID NO: 2, an insertion of AS at position 793 of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, a substitution of C477R of SEQ ID NO: 2, a substitution of C477K of SEQ ID NO: 2, a substitution of C479A of SEQ ID NO: 2, a substitution of C479L of SEQ ID NO: 2, a substitution of I55F of SEQ ID NO: 2, a substitution of K210R of SEQ ID NO: 2, a substitution of C233S of SEQ ID NO: 2, a substitution of D23 IN of SEQ ID NO: 2, a substitution of Q338E of SEQ ID NO: 2, a substitution of Q338R of SEQ ID NO: 2, a substitution of L379R of SEQ ID NO: 2, a substitution of K390R of SEQ ID NO: 2, a substitution of L481Q of SEQ ID NO: 2, a substitution of F495S of SEQ ID NO:2, a substitution of D600N of SEQ ID NO: 2, a substitution of T886K of SEQ ID NO: 2, a substitution of A739V of SEQ ID NO: 2, a substitution of K460N of SEQ ID NO: 2, a substitution of I199F of SEQ ID NO: 2, a substitution of G492P of SEQ ID NO: 2, a substitution of T153I of SEQ ID NO: 2, a substitution of R591I of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, an insertion of AS at position 796 of SEQ ID NO:2, an insertion of L at position 889 of SEQ ID NO: 2, a substitution of E121D of SEQ ID NO: 2, a substitution of S270W of SEQ ID NO: 2, a substitution of E712Q of SEQ ID NO: 2, a substitution of K942Q of SEQ ID NO: 2, a substitution of E552K of SEQ ID NO:2, a substitution of K25Q of SEQ ID NO: 2, a substitution of N47D of SEQ ID NO: 2, an insertion of T at position 696 of SEQ ID NO: 2, a substitution of L685I of SEQ ID NO: 2, a substitution of N880D of SEQ ID NO: 2, a substitution of Q102R of SEQ ID NO: 2, a substitution of M734K of SEQ ID NO: 2, a substitution of A724S of SEQ ID NO: 2, a substitution of T704K of SEQ ID NO: 2, a substitution of P224K of SEQ ID NO: 2, a substitution of K25R of SEQ ID NO: 2, a substitution of M29E of SEQ ID NO: 2, a substitution of H152D of SEQ ID NO: 2, a substitution of S219R of SEQ ID NO: 2, a substitution of E475K of SEQ ID NO: 2, a substitution of G226R of SEQ ID NO: 2, a substitution of A377K of SEQ ID NO: 2, a substitution of E480K of SEQ ID NO: 2, a substitution of K416E of SEQ ID NO: 2, a substitution of H164R of SEQ ID NO: 2, a substitution of K767R of SEQ ID NO: 2, a substitution of I7F of SEQ ID NO: 2, a substitution of M29R of SEQ ID NO: 2, a substitution of H435R of SEQ ID NO: 2, a substitution of E385Q of SEQ ID NO: 2, a substitution of E385K of SEQ ID NO: 2, a substitution of I279F of SEQ ID NO: 2, a substitution of D489S of SEQ ID NO: 2, a substitution of D732N of SEQ ID NO: 2, a substitution of A739T of SEQ ID NO: 2, a substitution of W885R of SEQ ID NO: 2, a substitution of E53K of SEQ ID NO: 2, a substitution of A238T of SEQ ID NO: 2, a substitution of P283Q of SEQ ID NO: 2, a substitution of E292K of SEQ ID NO: 2, a substitution of Q628E of SEQ ID NO: 2, a substitution of R388Q of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO: 2, a substitution of L792K of SEQ ID NO: 2, a substitution of L792E of SEQ ID NO: 2, a substitution of M779N of SEQ ID NO: 2, a substitution of G27D of SEQ ID NO: 2, a substitution of K955R of SEQ ID NO: 2, a substitution of S867R of SEQ ID NO: 2, a substitution of R693I of SEQ ID NO: 2, a substitution of F189Y of SEQ ID NO: 2, a substitution of V635M of SEQ ID NO: 2, a substitution of F399L of SEQ ID NO: 2, a substitution of E498K of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO: 2, a substitution of V254G of SEQ ID NO: 2, a substitution of P793S of SEQ ID NO: 2, a substitution of K188E of SEQ ID NO: 2, a substitution of QT945KI of SEQ ID NO: 2, a substitution of T620P of SEQ ID NO: 2, a substitution of T946P of SEQ ID NO: 2, a substitution of TT949PP of SEQ ID NO: 2, a substitution of N952T of SEQ ID NO: 2, a substitution of K682E of SEQ ID NO: 2, a substitution of K975R of SEQ ID NO: 2, a substitution of L212P of SEQ ID NO: 2, a substitution of E292R of SEQ ID NO: 2, a substitution of I303K of SEQ ID NO: 2, a substitution of C349E of SEQ ID NO: 2, a substitution of E385P of SEQ ID NO: 2, a substitution of E386N of SEQ ID NO: 2, a substitution of D387K of SEQ ID NO: 2, a substitution of L404K of SEQ ID NO: 2, a substitution of E466H of SEQ ID NO: 2, a substitution of C477Q of SEQ ID NO: 2, a substitution of C477H of SEQ ID NO: 2, a substitution of C479A of SEQ ID NO: 2, a substitution of D659H of SEQ ID NO: 2, a substitution of T806V of SEQ ID NO: 2, a substitution of K808S of SEQ ID NO: 2, an insertion of AS at position 797 of SEQ ID NO: 2, a substitution of V959M of SEQ ID NO: 2, a substitution of K975Q of SEQ ID NO: 2, a substitution of W974G of SEQ ID NO: 2, a substitution of A708Q of SEQ ID NO: 2, a substitution of V711K of SEQ ID NO: 2, a substitution of D733T of SEQ ID NO: 2, a substitution of L742W of SEQ ID NO: 2, a substitution of V747K of SEQ ID NO: 2, a substitution of F755M of SEQ ID NO: 2, a substitution of M771A of SEQ ID NO: 2, a substitution of M771Q of SEQ ID NO: 2, a substitution of W782Q of SEQ ID NO: 2, a substitution of G791F, of SEQ ID NO: 2, a substitution of L792D of SEQ ID NO: 2, a substitution of L792K of SEQ ID NO: 2, a substitution of P793Q of SEQ ID NO: 2, a substitution of P793G of SEQ ID NO: 2, a substitution of Q804A of SEQ ID NO: 2, a substitution of Y966N of SEQ ID NO: 2, a substitution of Y723N of SEQ ID NO: 2, a substitution of Y857R of SEQ ID NO: 2, a substitution of S890R of SEQ ID NO: 2, a substitution of S932M of SEQ ID NO: 2, a substitution of L897M of SEQ ID NO: 2, a substitution of R624G of SEQ ID NO: 2, a substitution of S603G of SEQ ID NO: 2, a substitution of N737S of SEQ ID NO: 2, a substitution of L307K of SEQ ID NO: 2, a substitution of I658V of SEQ ID NO: 2, an insertion of PT at position 688 of SEQ ID NO: 2, an insertion of SA at position 794 of SEQ ID NO: 2, a substitution of S877R of SEQ ID NO: 2, a substitution of N580T of SEQ ID NO: 2, a substitution of V335G of SEQ ID NO: 2, a substitution of T620S of SEQ ID NO: 2, a substitution of W345G of SEQ ID NO: 2, a substitution of T280S of SEQ ID NO: 2, a substitution of L406P of SEQ ID NO: 2, a substitution of A612D of SEQ ID NO: 2, a substitution of A751S of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO: 2, a substitution of V351M of SEQ ID NO: 2, a substitution of K210N of SEQ ID NO: 2, a substitution of D40A of SEQ ID NO: 2, a substitution of E773G of SEQ ID NO: 2, a substitution of H207L of SEQ ID NO: 2, a substitution of T62A SEQ ID NO: 2, a substitution of T287P of SEQ ID NO: 2, a substitution of T832A of SEQ ID NO: 2, a substitution of A893S of SEQ ID NO: 2, an insertion of V at position 14 of SEQ ID NO: 2, an insertion of AG at position 13 of SEQ ID NO: 2, a substitution of R11V of SEQ ID NO: 2, a substitution of R12N of SEQ ID NO: 2, a substitution of R13H of SEQ ID NO: 2, an insertion of Y at position 13 of SEQ ID NO: 2, a substitution of R12L of SEQ ID NO: 2, an insertion of Q at position 13 of SEQ ID NO: 2, an substitution of V15S of SEQ ID NO: 2 and an insertion of D at position 17 of SEQ ID NO: 2. In some embodiments, the at least two amino acid changes to a reference CasX protein are selected from the amino acid changes disclosed in the sequences of Table 3. In some embodiments, a CasX variant comprises any combination of the foregoing embodiments of this paragraph.

In some embodiments, a CasX variant protein comprises more than one substitution, insertion and/or deletion of a reference CasX protein amino acid sequence. In some embodiments, the reference CasX protein comprises or consists essentially of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of S794R and a substitution of Y797L of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of K416E and a substitution of A708K of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K and a deletion of P793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a deletion of P793 and an insertion of AS at position 795 SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of Q367K and a substitution of I425S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K, a deletion of P position 793 and a substitution A793V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of Q338R and a substitution of A339E of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of Q338R and a substitution of A339K of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of S507G and a substitution of G508R of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position of 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of 708K, a deletion of P at position 793 and a substitution of D489S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of G791M of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of 708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D489S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of G791M of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of T620P of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K, a deletion of P at position 793 and a substitution of E386S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of E386R, a substitution of F399L and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of R5811 and A739V of SEQ ID NO: 2. In some embodiments, a CasX variant comprises any combination of the foregoing embodiments of this paragraph.

In some embodiments, a CasX variant protein comprises more than one substitution, insertion and/or deletion of a reference CasX protein amino acid sequence. In some embodiments, a CasX variant protein comprises a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of T620P of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of M771A of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX variant comprises any combination of the foregoing embodiments of this paragraph.

In some embodiments, a CasX variant protein comprises a substitution of W782Q of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of M771Q of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of R458I and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D489S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of V711K of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K, a substitution of P at position 793 and a substitution of E386S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L792D of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of G791F of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K and a substitution of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L2491 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of V747K of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of F755M. In some embodiments, a CasX variant comprises any combination of the foregoing embodiments of this paragraph.

In some embodiments, a CasX variant protein comprises at least one modification compared to the reference CasX sequence of SEQ ID NO: 2, wherein the at least one modification is selected from one or more of an amino acid substitution of L379R; an amino acid substitution of A708K; an amino acid substitution of T620P; an amino acid substitution of E385P; an amino acid substitution of Y857R; an amino acid substitution of I658V; an amino acid substitution of F399L; an amino acid substitution of Q252K; an amino acid substitution of L404K; and an amino acid deletion of [P793]. In other embodiments, a CasX variant protein comprises any combination of the foregoing substitutions or deletions compared to the reference CasX sequence of SEQ ID NO: 2. In other embodiments, the CasX variant protein can, in addition to the foregoing substitutions or deletions, further comprise a substitution of an NTSB and/or a helical Ib domain from the reference CasX of SEQ ID NO: 1.

In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520 and 3540-3549.

In some embodiments, a CasX variant comprises one or modifications to any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, a CasX variant comprises one or modifications to any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, a CasX variant comprises one or modifications to any one of SEQ ID NOS: 3498-3501, 3505-3520 and 3540-3549.

In some embodiments, the CasX variant protein comprises between 400 and 2000 amino acids, between 500 and 1500 amino acids, between 700 and 1200 amino acids, between 800 and 1100 amino acids or between 900 and 1000 amino acids.

In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form a channel in which gNA:target DNA complexing occurs. In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous residues that form an interface which binds with the gNA. For example, in some embodiments of a reference CasX protein, the helical I, helical II and OBD domains all contact or are in proximity to the gNA:target DNA complex, and one or more modifications to non-contiguous residues within any of these domains may improve function of the CasX variant protein.

In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form a channel which binds with the non-target strand DNA. For example, a CasX variant protein can comprise one or more modifications to non-contiguous residues of the NTSBD. In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form an interface which binds with the PAM. For example, a CasX variant protein can comprise one or more modifications to non-contiguous residues of the helical I domain or OBD. In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous surface-exposed residues. As used herein, “surface-exposed residues” refers to amino acids on the surface of the CasX protein, or amino acids in which at least a portion of the amino acid, such as the backbone or a part of the side chain is on the surface of the protein. Surface exposed residues of cellular proteins such as CasX, which are exposed to an aqueous intracellular environment, are frequently selected from positively charged hydrophilic amino acids, for example arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Thus, for example, in some embodiments of the variants provided herein, a region of surface exposed residues comprises one or more insertions, deletions, or substitutions compared to a reference CasX protein. In some embodiments, one or more positively charged residues are substituted for one or more other positively charged residues, or negatively charged residues, or uncharged residues, or any combinations thereof. In some embodiments, one or more amino acids residues for substitution are near bound nucleic acid, for example residues in the RuvC domain or helical I domain that contact target DNA, or residues in the OBD or helical II domain that bind the gNA, can be substituted for one or more positively charged or polar amino acids.

In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form a core through hydrophobic packing in a domain of the reference CasX protein. Without wishing to be bound by any theory, regions that form cores through hydrophobic packing are rich in hydrophobic amino acids such as valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and cysteine. For example, in some reference CasX proteins, RuvC domains comprise a hydrophobic pocket adjacent to the active site. In some embodiments, between 2 to 15 residues of the region are charged, polar, or base-stacking. Charged amino acids (sometimes referred to herein as residues) may include, for example, arginine, lysine, aspartic acid, and glutamic acid, and the side chains of these amino acids may form salt bridges provided a bridge partner is also present (see FIG. 14). Polar amino acids may include, for example, glutamine, asparagine, histidine, serine, threonine, tyrosine, and cysteine. Polar amino acids can, in some embodiments, form hydrogen bonds as proton donors or acceptors, depending on the identity of their side chains. As used herein, “base-stacking” includes the interaction of aromatic side chains of an amino acid residue (such as tryptophan, tyrosine, phenylalanine, or histidine) with stacked nucleotide bases in a nucleic acid. Any modification to a region of non-contiguous amino acids that are in close spatial proximity to form a functional part of the CasX variant protein is envisaged as within the scope of the disclosure.

i. CasX Variant Proteins with Domains from Multiple Source Proteins

In certain embodiments, the disclosure provides a chimeric CasX protein comprising protein domains from two or more different CasX proteins, such as two or more naturally occurring CasX proteins, or two or more CasX variant protein sequences as described herein. As used herein, a “chimeric CasX protein” refers to a CasX containing at least two domains isolated or derived from different sources, such as two naturally occurring proteins, which may, in some embodiments, be isolated from different species. For example, in some embodiments, a chimeric CasX protein comprises a first domain from a first CasX protein and a second domain from a second, different CasX protein. In some embodiments, the first domain can be selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains. In some embodiments, the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains with the second domain being different from the foregoing first domain. For example, a chimeric CasX protein may comprise an NTSB, TSL, helical I, helical II, OBD domains from a CasX protein of SEQ ID NO: 2, and a RuvC domain from a CasX protein of SEQ ID NO: 1, or vice versa. As a further example, a chimeric CasX protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from CasX protein of SEQ ID NO: 2, and a helical I domain from a CasX protein of SEQ ID NO: 1, or vice versa. Thus, in certain embodiments, a chimeric CasX protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from a first CasX protein, and a helical I domain from a second CasX protein. In some embodiments of the chimeric CasX proteins, the domains of the first CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the domains of the second CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the first and second CasX proteins are not the same. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 2. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 2 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. In some embodiments, the CasX variant is selected of group consisting of CasX variants with sequences of SEQ ID NO: 328, SEQ ID NO: 3540, SEQ ID NO: 4413, SEQ ID NO: 4414, SEQ ID NO: 4415, SEQ ID NO: 329, SEQ ID NO: 3541, SEQ ID NO: 330, SEQ ID NO: 3542, SEQ ID NO: 331, SEQ ID NO: 3543, SEQ ID NO: 332, SEQ ID NO: 3544, SEQ ID NO: 333, SEQ ID NO: 3545, SEQ ID NO: 334, SEQ ID NO: 3546, SEQ ID NO: 335, SEQ ID NO: 3547, SEQ ID NO: 336 and SEQ ID NO: 3548. In some embodiments, the CasX variant comprises one or more additional modifications to any one of SEQ ID NO: 328, SEQ ID NO: 3540, SEQ ID NO: 4413, SEQ ID NO: 4414, SEQ ID NO: 4415, SEQ ID NO: 329, SEQ ID NO: 3541, SEQ ID NO: 330, SEQ ID NO: 3542, SEQ ID NO: 331, SEQ ID NO: 3543, SEQ ID NO: 332, SEQ ID NO: 3544, SEQ ID NO: 333, SEQ ID NO: 3545, SEQ ID NO: 334, SEQ ID NO: 3546, SEQ ID NO: 335, SEQ ID NO: 3547, SEQ ID NO: 336 or SEQ ID NO: 3548. In some embodiments, the one or more additional modifications comprises an insertion, substitution or deletion as described herein.

In some embodiments, a CasX variant protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second, different CasX protein. As used herein, a “chimeric domain” refers to a domain containing at least two parts isolated or derived from different sources, such as two naturally occurring proteins or portions of domains from two reference CasX proteins. The at least one chimeric domain can be any of the NTSB, TSL, helical I, helical II, OBD or RuvC domains as described herein. In some embodiments, the first portion of a CasX domain comprises a sequence of SEQ ID NO: 1 and the second portion of a CasX domain comprises a sequence of SEQ ID NO: 2. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ ID NO: 1 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ ID NO: 2 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the at least one chimeric domain comprises a chimeric RuvC domain. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 661 to 824 of SEQ ID NO: 1 and amino acids 922 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ ID NO: 1. In some embodiments, a CasX protein comprises a first domain from a first CasX protein and a second domain from a second CasX protein, and at least one chimeric domain comprising at least two parts isolated from different CasX proteins using the approach of the embodiments described in this paragraph. In the foregoing embodiments, the chimeric CasX proteins having domains or portions of domains derived from SEQ ID NOS: 1, 2 and 3, can further comprise amino acid insertions, deletions, or substitutions of any of the embodiments disclosed herein.

In some embodiments, a CasX variant protein comprises a sequence set forth in Tables 3, 8, 9, 10 or 12. In other embodiments, a CasX variant protein comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to a sequence set forth in Tables 3, 8, 9, 10 or 12. In other embodiments, a CasX variant protein comprises a sequence set forth in Table 3, and further comprises one or more NLS disclosed herein on either the N-terminus, the C-terminus, or both. It will be understood that in some cases, the N-terminal methionine of the CasX variants of the Tables is removed from the expressed CasX variant during post-translational modification.

TABLE 3 CasX Variant Sequences Description* Amino Acid Sequence TSL, Helical I, Helical II OBD and RuvC domains from SEQ ID NO: 2 and an NTSB SEQ ID NO: 247 domain from SEQ ID NO: 1 NTSB, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 2 and a TSL SEQ ID NO: 248 domain from SEQ ID NO: 1. TSL, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 1 and an NTSB SEQ ID NO: 249 domain from SEQ ID NO: 2 NTSB, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 1 and an TSL SEQ ID NO: 250 domain from SEQ ID NO: 2. NTSB, TSL, Helical I, Helical II and OBD domains SEQ ID NO: 2 and an exogenous SEQ ID NO: 251 RuvC domain or a portion thereof from a second CasX protein. No description SEQ ID NO: 252 NTSB, TSL, Helical II, OBD and RuvC domains from SEQ ID NO: 2 and a Helical I SEQ ID NO: 253 domain from SEQ ID NO: I NTSB, TSL, Helical I, OBD and RuvC domains from SEQ ID NO: 2 and a Helical II SEQ ID NO: 254 domain from SEQ ID NO: 1 NTSB, TSL, Helical I, Helical II and RuvC domains from a first CasX protein and an SEQ ID NO: 255 exogenous OBD or a part thereof from a second CasX protein No description SEQ ID NO: 256 Nodescription SEQ ID NO: 257 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 258 P at position 793 and a substitution of T620P of SEQ ID NO: 2 substitution of M77 IA of SEQ. ID NO: 2. SEQ ID NO: 259 substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a SEQ ID NO: 260 substitution of D732N of SEQ ID NO: 2. substitution of W782Q of SEQ ID NO: 2. SEQ ID NO: 261 substitution of M771Q of SEQ ID NO: 2 SEQ ID NO: 262 substitution of R458I and a substitution of A739V of SEQ ID NO: 2. SEQ ID NO: 263 L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of SEQ ID NO: 264 M771N of SEQ ID NO: 2 substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a SEQ ID NO: 265 stibstitution of A739T of SEQ ID NO: 2 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 266 P at position 793 and a substitution of D4895 of SEQ ID NO: 2. substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 267 P at position 793 and a substitution of D732N of SEQ, ID NO: 2. substitution of V711K of SEQ ID NO: 2. SEQ ID NO: 268 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 269 P at position 793 and a substitution of Y797L of SEQ ID NO: 2. 119, substitution of L379R, a substitution of A708K and a deletion of P at position 793 SEQ ID NO: 270 of SEQ ID NO: 2. substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 271 P at position 793 and a substitution of M771N of SEQ ID NO: 2. substitution of A708K, a deletion of P at position 793 and a substitution of E3865 of SEQ ID NO: 272 SEQ ID NO: 2. substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion SEQ ID NO: 273 of P at position 793 of SEQ ID NO: 2. substitution of L792D of SEQ ID NO: 2. SEQ ID NO: 274 substitution of G791F of SEQ ID NO: 2. SEQ ID NO: 275 substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 276 SEQ ID NO: 2. substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a SEQ ID NO: 277 substitution of A739V of SEQ ID NO: 2. substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 278 SEQ ID NO: 2. substitution of L249R and a substitution of M771N of SEQ ID NO: 2. SEQ ID NO: 279 substitution of V747K of SEQ ID NO: 2. SEQ ID NO: 280 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 281 P at position 793 and a substitution of M779N of SEQ ID NO: 2. L379R, F755M SEQ ID NO: 282 429, L379R, A708K, P793_, Y857R SEQ ID NO: 283 430, L379R, A708K, P793_, Y857R, I658V SEQ ID NO: 284 431, L379R, A708K, P793_, Y857R, I658V, E38614 SEQ ID NO: 285 432, L379R, A708K, P793_, Y857R, I658V, L404K SEQ ID NO: 286 433, L379R, A708K, P793_, Y857R, I658V, {circumflex over ( )}V192 SEQ ID NO: 287 434, L379R, A708K, P793_, Y857R, I658V, L404K, E386N SEQ ID NO: 288 435, L379R, A708K, P793_, Y857R, I658V, F399L SEQ ID NO: 289 436, L379R, A708K, P793_, Y857R, I658V, F399L, E386N SEQ ID NO: 290 437, L379R, A708K, P793_, Y857R, I658V, F399L, C4775 SEQ ID NO: 291 438, L379R, A708K, P793_, Y857R, I658V, F399L, L404K SEQ ID NO: 292 439, L379R, A708K, P793_, Y857R, I658V, F399L, E386N, C4775, L404K SEQ. ID NO: 293 440, L379R, A708K, P793_, Y857R, I658V, F399L, Y797L SEQ ID NO: 294 441, L379R, A708K, P793_ , Y857R, I658V, F399L, Y797L, E386N SEQ ID NO: 295 442, L379R, A708K, P793_, Y857R, I658V, F399L, Y797L, E386N, C4775, L404K SEQ ID NO: 296 443, L379R, A708K, P793_, Y857R, I658V, Y797L SEQ ID NO: 297 444, L379R, A708K, P793_, Y857R, I651W, Y797L, L404K SEQ ID NO: 298 445, L379R, A708K, P793_, Y857R, I651W, Y797L, E386N SEQ ID NO: 299 446, L379R, A708K, P793_, Y857R, I658V, Y797L, E386N, C4775, L404K SEQ ID NO: 300 447, L379R, A708K, P793_, Y857R, E386N SEQ ID NO: 301 448, L379R, A708K, P793_, Y857R, E386N, L404K SEQ ID NO: 302 449, L379R, A708K, P793_, D732N, E385P, Y857R SEQ ID NO: 303 450, L379R, A708K, P793_, D732N, E385P, Y857R, I651W SEQ ID NO: 304 451, L379R, A708K, P793_, D732N, E385P, Y857R, I651W, F399L SEQ ID NO: 305 452, L379R, A708K, P793_, D732N, E385P, Y857R, I658V, E386N SEQ ID NO: 306 453, L379R, A708K, P793_, D732N, E385P, Y857R, I658V, L404K SEQ ID NO: 307 454, L379R, A708K, P793_, T620P, E385P, Y857R, Q252K SEQ ID NO: 308 455, L379R, A708K, P793_, T620P, E385P, Y857R, I651W, Q252K SEQ ID NO: 309 456, L379R, A708K, P793_, T620P, E385P, Y857R, I651W, E386N, Q252K SEQ ID NO: 310 457, L379R, A708K, P793_, T620P, E385P, Y857R, I658V, F399L, Q252K SEQ ID NO: 311 458, L379R, A708K, P793_, T620P, E385P, Y85712, I651W, L404K, Q252K SEQ ID NO: 312 459, L379R, A708K, P793_, T620P, Y857R, I658V, E386N SEQ ID NO: 313 460, L379R, A708K, P793_ , T620P, E385P, Q252K SEQ ID NO: 314 278 SEQ ID NO: 315 279 SEQ ID NO: 316 280 SEQ ID NO: 317 285 SEQ ID NO: 318 286 SEQ ID NO: 319 287 SEQ ID NO: 320 288 SEQ ID NO: 321 290 SEQ ID NO: 322 291 SEQ ID NO: 323 293 SEQ ID NO: 324 300 SEQ ID NO: 325 492 SEQ ID NO: 326 493 SEQ ID NO: 327 387, NTSB swap from SEQ ID NO: 1 SEQ ID NO: 328 395, Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 329 485, Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 330 486, Helical 1B swap from SEQ 1D NO: 1 SEQ ID NO: 331 487, Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 332 488, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 333 489, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 334 490, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 335 491, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 336 494, NTSB swap from SEQ ID NO: 1 SEQ ID NO: 337 328, S867G  SEQ ID NO: 4412 388, L379R + A708K + [P793] + X1 Helical2 swap  SEQ ID NO: 4413 389, L379R + A708K + [P793] + X1 RuvC1 swap  SEQ ID NO: 4414 390, L379R + A708K + [P793] + X1 RuvC2 swap  SEQ ID NO: 4415 *Strain indicated numerically; changes, where indicated, are relative to SEQ ID NO: 2

In some embodiments, the CasX variant protein has one or more improved characteristics when compared to a reference CasX protein, for example a reference protein of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, an improved characteristic of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference protein. In some embodiments, an improved characteristic of the CasX variant is at least about 1.1 to about 10,000-fold improved, at least about 1.1 to about 1,000-fold improved, at least about 1.1 to about 500-fold improved, at least about 1.1 to about 400-fold improved, at least about 1.1 to about 300-fold improved, at least about 1.1 to about 200-fold improved, at least about 1.1 to about 100-fold improved, at least about 1.1 to about 50-fold improved, at least about 1.1 to about 40-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 10-fold improved, at least about 1.1 to about 9-fold improved, at least about 1.1 to about 8-fold improved, at least about 1.1 to about 7-fold improved, at least about 1.1 to about 6-fold improved, at least about 1.1 to about 5-fold improved, at least about 1.1 to about 4-fold improved, at least about 1.1 to about 3-fold improved, at least about 1.1 to about 2-fold improved, at least about 1.1 to about 1.5-fold improved, at least about 1.5 to about 3-fold improved, at least about 1.5 to about 4-fold improved, at least about 1.5 to about 5-fold improved, at least about 1.5 to about 10-fold improved, at least about 5 to about 10-fold improved, at least about 10 to about 20-fold improved, at least 10 to about 30-fold improved, at least 10 to about 50-fold improved or at least 10 to about 100-fold improved than the reference CasX protein. In some embodiments, an improved characteristic of the CasX variant is at least about 10 to about 1000-fold improved relative to the reference CasX protein.

In some embodiments, the one or more improved characteristics of the CasX variant protein is at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 250, at least about 500, or at least about 1000, at least about 5,000, at least about 10,000, or at least about 100,000-fold improved relative to a reference CasX protein. In some embodiments, an improved characteristics of the CasX variant protein is at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 at least about 100, at least about 500, at least about 1,000, at least about 10,000, or at least about 100,000-fold improved relative to a reference CasX protein. In other cases, the one or more improved characteristics of the CasX variant is about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to the reference CasX of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In other cases, the one or more improved characteristics of the CasX variant is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold or more improved relative to the reference CasX of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. Exemplary characteristics that can be improved in CasX variant proteins relative to the same characteristics in reference CasX proteins include, but are not limited to, improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target DNA, improved ability to utilize a greater spectrum of PAM sequences in the editing and/or binding of target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved CasX:gNA RNA complex stability, improved protein solubility, improved CasX:gNA RNP complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics. In some embodiments, the variant comprises at least one improved characteristic. In other embodiments, the variant comprises at least two improved characteristics. In further embodiments, the variant comprises at least three improved characteristics. In some embodiments, the variant comprises at least four improved characteristics. In still further embodiments, the variant comprises at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or more improved characteristics. These improved characteristics are described in more detail below.

j. Protein Stability

In some embodiments, the disclosure provides a CasX variant protein with improved stability relative to a reference CasX protein. In some embodiments, improved stability of the CasX variant protein results in expression of a higher steady state of protein, which improves editing efficiency. In some embodiments, improved stability of the CasX variant protein results in a larger fraction of CasX protein that remains folded in a functional conformation and improves editing efficiency or improves purifiability for manufacturing purposes. As used herein, a “functional conformation” refers to a CasX protein that is in a conformation where the protein is capable of binding a gNA and target DNA. In embodiments wherein the CasX variant does not carry one or more mutations rendering it catalytically dead, the CasX variant is capable of cleaving, nicking, or otherwise modifying the target DNA. For example, a functional CasX variant can, in some embodiments, be used for gene-editing, and a functional conformation refers to an “editing-competent” conformation. In some exemplary embodiments, including those embodiments where the CasX variant protein results in a larger fraction of CasX protein that remains folded in a functional conformation, a lower concentration of CasX variant is needed for applications such as gene editing compared to a reference CasX protein. Thus, in some embodiments, the CasX variant with improved stability has improved efficiency compared to a reference CasX in one or more gene editing contexts.

In some embodiments, the disclosure provides a CasX variant protein having improved thermostability relative to a reference CasX protein. In some embodiments, the CasX variant protein has improved thermostability of the CasX variant protein at a particular temperature range. Without wishing to be bound by any theory, some reference CasX proteins natively function in organisms with niches in groundwater and sediment; thus, some reference CasX proteins may have evolved to exhibit optimal function at lower or higher temperatures that may be desirable for certain applications. For example, one application of CasX variant proteins is gene editing of mammalian cells, which is typically carried out at about 37° C. In some embodiments, a CasX variant protein as described herein has improved thermostability compared to a reference CasX protein at a temperature of at least 16° C., at least 18° C. at least 20° C., at least 22° C., at least 24° C., at least 26° C., at least 28° C., at least 30° C., at least 32° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., at least 42° C., at least 44° C., at least 46° C., at least 48° C., at least 50° C., at least 52° C., or greater. In some embodiments, a CasX variant protein has improved thermostability and functionality compared to a reference CasX protein that results in improved gene editing functionality, such as mammalian gene editing applications, which may include human gene editing applications.

In some embodiments, the disclosure provides a CasX variant protein having improved stability of the CasX variant protein:gNA RNP complex relative to the reference CasX protein:gNA complex such that the RNP remains in a functional form. Stability improvements can include increased thermostability, resistance to proteolytic degradation, enhanced pharmacokinetic properties, stability across a range of pH conditions, salt conditions, and tonicity. Improved stability of the complex may, in some embodiments, lead to improved editing efficiency.

In some embodiments, the disclosure provides a CasX variant protein having improved thermostability of the CasX variant protein:gNA complex relative to the reference CasX protein:gNA complex. In some embodiments, a CasX variant protein has improved thermostability relative to a reference CasX protein. In some embodiments, the CasX variant protein:gNA RNP complex has improved thermostability relative to a complex comprising a reference CasX protein at temperatures of at least 16° C., at least 18° C., at least 20° C., at least 22° C., at least 24° C., at least 26° C., at least 28° C., at least 30° C., at least 32° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C. at least 42° C., at least 44° C., at least 46° C., at least 48° C., at least 50° C., at least 52° C., or greater. In some embodiments, a CasX variant protein has improved thermostability of the CasX variant protein:gNA RNP complex compared to a reference CasX protein:gNA complex, which results in improved function for gene editing applications, such as mammalian gene editing applications, which may include human gene editing applications.

In some embodiments, the improved stability and/or thermostability of the CasX variant protein comprises faster folding kinetics of the CasX variant protein relative to a reference CasX protein, slower unfolding kinetics of the CasX variant protein relative to a reference CasX protein, a larger free energy release upon folding of the CasX variant protein relative to a reference CasX protein, a higher temperature at which 50% of the CasX variant protein is unfolded (Tm) relative to a reference CasX protein, or any combination thereof. These characteristics may be improved by a wide range of values; for example, at least 1.1, at least 1.5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, or at least a 10,000-fold improved, as compared to a reference CasX protein. In some embodiments, improved thermostability of the CasX variant protein comprises a higher Tm of the CasX variant protein relative to a reference CasX protein. In some embodiments, the Tm of the CasX variant protein is between about 20° C. to about 30° C., between about 30° C. to about 40° C., between about 40° C. to about 50° C., between about 50° C. to about 60° C., between about 60° C. to about 70° C., between about 70° C. to about 80° C., between about 80° C. to about 90° C. or between about 90° C. to about 100° C. Thermal stability is determined by measuring the “melting temperature” (Tm), which is defined as the temperature at which half of the molecules are denatured. Methods of measuring characteristics of protein stability such as Tm and the free energy of unfolding are known to persons of ordinary skill in the art, and can be measured using standard biochemical techniques in vitro. For example, Tm may be measured using Differential Scanning Calorimetry, a thermo-analytical technique in which the difference in the amount of heat required to increase the temperature of a sample and a reference is measured as a function of temperature (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). Alternatively, or in addition, CasX variant protein Tm may be measured using commercially available methods such as the ThermoFisher Protein Thermal Shift system. Alternatively, or in addition, circular dichroism may be used to measure the kinetics of folding and unfolding, as well as the Tm (Murray et al. (2002) J. Chromatogr Sci 40:343-9). Circular dichroism (CD) relies on the unequal absorption of left-handed and right-handed circularly polarized light by asymmetric molecules such as proteins. Certain structures of proteins, for example alpha-helices and beta-sheets, have characteristic CD spectra. Accordingly, in some embodiments, CD may be used to determine the secondary structure of a CasX variant protein.

In some embodiments, improved stability and/or thermostability of the CasX variant protein comprises improved folding kinetics of the CasX variant protein relative to a reference CasX protein. In some embodiments, folding kinetics of the CasX variant protein are improved relative to a reference CasX protein by at least about 5, at least about 10, at least about 50, at least about 100, at least about 500, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, or at least about a 10,000-fold improvement. In some embodiments, folding kinetics of the CasX variant protein are improved relative to a reference CasX protein by at least about 1 kJ/mol, at least about 5 kJ/mol, at least about 10 kJ/mol, at least about 20 kJ/mol, at least about 30 kJ/mol, at least about 40 kJ/mol, at least about 50 kJ/mol, at least about 60 kJ/mol, at least about 70 kJ/mol, at least about 80 kJ/mol, at least about 90 kJ/mol, at least about 100 kJ/mol, at least about 150 kJ/mol, at least about 200 kJ/mol, at least about 250 kJ/mol, at least about 300 kJ/mol, at least about 350 kJ/mol, at least about 400 kJ/mol, at least about 450 kJ/mol, or at least about 500 kJ/mol.

Exemplary amino acid changes that can increase the stability of a CasX variant protein relative to a reference CasX protein may include, but are not limited to, amino acid changes that increase the number of hydrogen bonds within the CasX variant protein, increase the number of disulfide bridges within the CasX variant protein, increase the number of salt bridges within the CasX variant protein, strengthen interactions between parts of the CasX variant protein, increase the buried hydrophobic surface area of the CasX variant protein, or any combinations thereof.

k. Protein Yield

In some embodiments, the disclosure provides a CasX variant protein having improved yield during expression and purification relative to a reference CasX protein. In some embodiments, the yield of CasX variant proteins purified from bacterial or eukaryotic host cells is improved relative to a reference CasX protein. In some embodiments, the bacterial host cells are Escherichia coli cells. In some embodiments, the eukaryotic cells are yeast, plant (e.g. tobacco), insect (e.g. Spodoptera frugiperda sf9 cells), mouse, rat, hamster, guinea pig, non-human primate, or human cells. In some embodiments, the eukaryotic host cells are mammalian cells, including, but not limited to HEK293 cells, HEK293T cells, HEK293-F cells, Lenti-X 293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, A549 cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS, W138 cells, MRC5 cells, HeLa, HT1080 cells, or CHO cells.

In some embodiments, improved yield of the CasX variant protein is achieved through codon optimization. Cells use 64 different codons, 61 of which encode the 20 standard amino acids, while another 3 function as stop codons. In some cases, a single amino acid is encoded by more than one codon. Different organisms exhibit bias towards use of different codons for the same naturally occurring amino acid. Therefore, the choice of codons in a protein, and matching codon choice to the organism in which the protein will be expressed, can, in some cases, significantly affect protein translation and therefore protein expression levels. In some embodiments, the CasX variant protein is encoded by a nucleic acid that has been codon optimized. In some embodiments, the nucleic acid encoding the CasX variant protein has been codon optimized for expression in a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. In some embodiments, the mammal cell is a mouse, a rat, a hamster, a guinea pig, a monkey, or a human. In some embodiments, the CasX variant protein is encoded by a nucleic acid that has been codon optimized for expression in a human cell. In some embodiments, the CasX variant protein is encoded by a nucleic acid from which nucleotide sequences that reduce translation rates in prokaryotes and eukaryotes have been removed. For example, runs of greater than three thymine residues in a row can reduce translation rates in certain organisms or internal polyadenylation signals can reduce translation.

In some embodiments, improvements in solubility and stability, as described herein, result in improved yield of the CasX variant protein relative to a reference CasX protein.

Improved protein yield during expression and purification can be evaluated by methods known in the art. For example, the amount of CasX variant protein can be determined by running the protein on an SDS-page gel, and comparing the CasX variant protein to a control whose amount or concentration is known in advance to determine an absolute level of protein. Alternatively, or in addition, a purified CasX variant protein can be run on an SDS-page gel next to a reference CasX protein undergoing the same purification process to determine relative improvements in CasX variant protein yield. Alternatively, or in addition, levels of protein can be measured using immunohistochemical methods such as Western blot or ELISA with an antibody to CasX, or by HPLC. For proteins in solution, concentration can be determined by measuring of the protein's intrinsic UV absorbance, or by methods which use protein-dependent color changes such as the Lowry assay, the Smith copper/bicinchoninic assay or the Bradford dye assay. Such methods can be used to calculate the total protein (such as, for example, total soluble protein) yield obtained by expression under certain conditions. This can be compared, for example, to the protein yield of a reference CasX protein under similar expression conditions.

l. Protein Solubility

In some embodiments, a CasX variant protein has improved solubility relative to a reference CasX protein. In some embodiments, a CasX variant protein has improved solubility of the CasX:gNA ribonucleoprotein complex variant relative to a ribonucleoprotein complex comprising a reference CasX protein.

In some embodiments, an improvement in protein solubility leads to higher yield of protein from protein purification techniques such as purification from E. coli. Improved solubility of CasX variant proteins may, in some embodiments, enable more efficient activity in cells, as a more soluble protein may be less likely to aggregate in cells. Protein aggregates can in certain embodiments be toxic or burdensome on cells, and, without wishing to be bound by any theory, increased solubility of a CasX variant protein may ameliorate this result of protein aggregation. Further, improved solubility of CasX variant proteins may allow for enhanced formulations permitting the delivery of a higher effective dose of functional protein, for example in a desired gene editing application. In some embodiments, improved solubility of a CasX variant protein relative to a reference CasX protein results in improved yield of the CasX variant protein during purification of at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 250, at least about 500, or at least about 1000-fold greater yield. In some embodiments, improved solubility of a CasX variant protein relative to a reference CasX protein improves activity of the CasX variant protein in cells by at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15-fold, or at least about 20-fold greater activity.

Methods of measuring CasX protein solubility, and improvements thereof in CasX variant proteins, will be readily apparent to the person of ordinary skill in the art. For example, CasX variant protein solubility can in some embodiments be measured by taking densitometry readings on a gel of the soluble fraction of lysed E. coli. Alternatively, or addition, improvements in CasX variant protein solubility can be measured by measuring the maintenance of soluble protein product through the course of a full protein purification, including the methods of the Examples. For example, soluble protein product can be measured at one or more steps of gel affinity purification, tag cleavage, cation exchange purification, running the protein on a size exclusion chromatography (SEC) column. In some embodiments, the densitometry of every band of protein on a gel is read after each step in the purification process. CasX variant proteins with improved solubility may, in some embodiments, maintain a higher concentration at one or more steps in the protein purification process when compared to the reference CasX protein, while an insoluble protein variant may be lost at one or more steps due to buffer exchanges, filtration steps, interactions with a purification column, and the like.

In some embodiments, improving the solubility of CasX variant proteins results in a higher yield in terms of mg/L of protein during protein purification when compared to a reference CasX protein.

In some embodiments, improving the solubility of CasX variant proteins enables a greater amount of editing events compared to a less soluble protein when assessed in editing assays such as the EGFP disruption assays described herein.

m. Affinity for the gNA

In some embodiments, a CasX variant protein has improved affinity for the gNA relative to a reference CasX protein, leading to the formation of the ribonucleoprotein complex. Increased affinity of the CasX variant protein for the gNA may, for example, result in a lower K_(d) for the generation of a RNP complex, which can, in some cases, result in a more stable ribonucleoprotein complex formation. In some embodiments, the K_(d) of a CasX variant protein for a gNA is increased relative to a reference CasX protein by a factor of at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100. In some embodiments, the CasX variant has about 1.1 to about 10-fold increased binding affinity to the gNA compared to the reference CasX protein of SEQ ID NO: 2.

In some embodiments, increased affinity of the CasX variant protein for the gNA results in increased stability of the ribonucleoprotein complex when delivered to mammalian cells, including in vivo delivery to a subject. This increased stability can affect the function and utility of the complex in the cells of a subject, as well as result in improved pharmacokinetic properties in blood, when delivered to a subject. In some embodiments, increased affinity of the CasX variant protein, and the resulting increased stability of the ribonucleoprotein complex, allows for a lower dose of the CasX variant protein to be delivered to the subject or cells while still having the desired activity, for example in vivo or in vitro gene editing. The increased ability to form RNP and keep them in stable form can be assessed using assays such as the in vitro cleavage assays described herein. In some embodiments, the CasX variants of the disclosure are able to achieve a K_(cleave) rate when complexed as an RNP that is at last 2-fold, at least 5-fold, or at least 10-fold higher compared to RNP of reference CasX.

In some embodiments, a higher affinity (tighter binding) of a CasX variant protein to a gNA allows for a greater amount of editing events when both the CasX variant protein and the gNA remain in an RNP complex. Increased editing events can be assessed using editing assays such as the EGFP disruption and in vitro cleavage assays described herein.

Without wishing to be bound by theory, in some embodiments amino acid changes in the helical I domain can increase the binding affinity of the CasX variant protein with the gNA targeting sequence, while changes in the helical II domain can increase the binding affinity of the CasX variant protein with the gNA scaffold stem loop, and changes in the oligonucleotide binding domain (OBD) increase the binding affinity of the CasX variant protein with the gNA triplex.

Methods of measuring CasX protein binding affinity for a gNA include in vitro methods using purified CasX protein and gNA. The binding affinity for reference CasX and variant proteins can be measured by fluorescence polarization if the gNA or CasX protein is tagged with a fluorophore. Alternatively, or in addition, binding affinity can be measured by biolayer interferometry, electrophoretic mobility shift assays (EMSAs), or filter binding. Additional standard techniques to quantify absolute affinities of RNA binding proteins such as the reference CasX and variant proteins of the disclosure for specific gNAs such as reference gNAs and variants thereof include, but are not limited to, isothermal calorimetry (ITC), and surface plasmon resonance (SPR), as well as the methods of the Examples.

n. Affinity for Target Nucleic Acid

In some embodiments, a CasX variant protein has improved binding affinity for a target nucleic acid relative to the affinity of a reference CasX protein for a target nucleic acid. CasX variants with higher affinity for their target nucleic acid may, in some embodiments, cleave the target nucleic acid sequence more rapidly than a reference CasX protein that does not have increased affinity for the target nucleic acid.

In some embodiments, the improved affinity for the target nucleic acid comprises improved affinity for the target sequence or protospacer sequence of the target nucleic acid, improved affinity for the PAM sequence, an improved ability to search DNA for the target sequence, or any combinations thereof. Without wishing to be bound by theory, it is thought that CRISPR/Cas system proteins such as CasX may find their target sequences by one-dimension diffusion along a DNA molecule. The process is thought to include (1) binding of the ribonucleoprotein to the DNA molecule followed by (2) stalling at the target sequence, either of which may be, in some embodiments, affected by improved affinity of CasX proteins for a target nucleic acid sequence, thereby improving function of the CasX variant protein compared to a reference CasX protein.

In some embodiments, a CasX variant protein with improved target nucleic acid affinity has increased overall affinity for DNA. In some embodiments, a CasX variant protein with improved target nucleic acid affinity has increased affinity for or the ability to utilize specific PAM sequences other than the canonical TTC PAM recognized by the reference CasX protein of SEQ ID NO: 2, including PAM sequences selected from the group consisting of TTC, ATC, GTC, and CTC, thereby increasing the amount of target DNA that can be edited compared to wild-type CasX nucleases. Without wishing to be bound by theory, it is possible that these protein variants may interact more strongly with DNA overall and may have an increased ability to access and edit sequences within the target DNA due to the ability to utilize additional PAM sequences beyond those of wild-type reference CasX, thereby allowing for a more efficient search process of the CasX protein for the target sequence. A higher overall affinity for DNA also, in some embodiments, can increase the frequency at which a CasX protein can effectively start and finish a binding and unwinding step, thereby facilitating target strand invasion and R-loop formation, and ultimately the cleavage of a target nucleic acid sequence.

Without wishing to be bound by theory, it is possible that amino acid changes in the NTSBD that increase the efficiency of unwinding, or capture, of a non-target DNA strand in the unwound state, can increase the affinity of CasX variant proteins for target DNA. Alternatively, or in addition, amino acid changes in the NTSBD that increase the ability of the NTSBD to stabilize DNA during unwinding can increase the affinity of CasX variant proteins for target DNA. Alternatively, or in addition, amino acid changes in the OBD may increase the affinity of CasX variant protein binding to the protospacer adjacent motif (PAM), thereby increasing affinity of the CasX variant protein for target nucleic acid. Alternatively, or in addition, amino acid changes in the Helical I and/or II, RuvC and TSL domains that increase the affinity of the CasX variant protein for the target nucleic acid strand can increase the affinity of the CasX variant protein for target nucleic acid.

In some embodiments, binding affinity of a CasX variant protein of the disclosure for a target nucleic acid molecule is increased relative to a reference CasX protein by a factor of at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100. In some embodiments, the CasX variant protein has about 1.1 to about 100-fold increased binding affinity to the target nucleic acid compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, a CasX variant protein has improved binding affinity for the non-target strand of the target nucleic acid. As used herein, the term “non-target strand” refers to the strand of the DNA target nucleic acid sequence that does not form Watson and Crick base pairs with the targeting sequence in the gNA, and is complementary to the target DNA strand. In some embodiments, the CasX variant protein has about 1.1 to about 100-fold increased binding affinity to the non-target stand of the target nucleic acid compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Methods of measuring CasX protein (such as reference or variant) affinity for a target and/or non-target nucleic acid molecule may include electrophoretic mobility shift assays (EMSAs), filter binding, isothermal calorimetry (ITC), and surface plasmon resonance (SPR), fluorescence polarization and biolayer interferometry (BLI). Further methods of measuring CasX protein affinity for a target include in vitro biochemical assays that measure DNA cleavage events over time.

o. Improved Specificity for a Target Site

In some embodiments, a CasX variant protein has improved specificity for a target nucleic acid sequence relative to a reference CasX protein. As used herein. “specificity,” sometimes referred to as “target specificity,” refers to the degree to which a CRISPR/Cas system ribonucleoprotein complex cleaves off-target sequences that are similar, but not identical to the target nucleic acid sequence; e.g., a CasX variant RNP with a higher degree of specificity would exhibit reduced off-target cleavage of sequences relative to a reference CasX protein. The specificity, and the reduction of potentially deleterious off-target effects, of CRISPR/Cas system proteins can be vitally important in order to achieve an acceptable therapeutic index for use in mammalian subjects.

In some embodiments, a CasX variant protein has improved specificity for a target site within the target sequence that is complementary to the targeting sequence of the gNA. Without wishing to be bound by theory, it is possible that amino acid changes in the helical I and II domains that increase the specificity of the CasX variant protein for the target nucleic acid strand can increase the specificity of the CasX variant protein for the target nucleic acid overall. In some embodiments, amino acid changes that increase specificity of CasX variant proteins for target nucleic acid may also result in decreased affinity of CasX variant proteins for DNA.

Methods of testing CasX protein (such as variant or reference) target specificity may include guide and Circularization for In vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq), or similar methods. In brief, in CIRCLE-seq techniques, genomic DNA is sheared and circularized by ligation of stem-loop adapters, which are nicked in the stem-loop regions to expose 4 nucleotide palindromic overhangs. This is followed by intramolecular ligation and degradation of remaining linear DNA. Circular DNA molecules containing a CasX cleavage site are subsequently linearized with CasX, and adapter adapters are ligated to the exposed ends followed by high-throughput sequencing to generate paired end reads that contain information about the off-target site. Additional assays that can be used to detect off-target events, and therefore CasX protein specificity include assays used to detect and quantify indels (insertions and deletions) formed at those selected off-target sites such as mismatch-detection nuclease assays and next generation sequencing (NGS). Exemplary mismatch-detection assays include nuclease assays, in which genomic DNA from cells treated with CasX and sgNA is PCR amplified, denatured and rehybridized to form hetero-duplex DNA, containing one wild type strand and one strand with an indel. Mismatches are recognized and cleaved by mismatch detection nucleases, such as Surveyor nuclease or T7 endonuclease 1.

p. Protospacer and PAM Sequences

Herein, the protospacer is defined as the DNA sequence complementary to the targeting sequence of the guide RNA and the DNA complementary to that sequence, referred to as the target strand and non-target strand, respectively. As used herein, the PAM is a nucleotide sequence proximal to the protospacer that, in conjunction with the targeting sequence of the gNA, helps the orientation and positioning of the CasX for the potential cleavage of the protospacer strand(s).

PAM sequences may be degenerate, and specific RNP constructs may have different preferred and tolerated PAM sequences that support different efficiencies of cleavage. Following convention, unless stated otherwise, the disclosure refers to both the PAM and the protospacer sequence and their directionality according to the orientation of the non-target strand. This does not imply that the PAM sequence of the non-target strand, rather than the target strand, is determinative of cleavage or mechanistically involved in target recognition. For example, when reference is to a TTC PAM, it may in fact be the complementary GAA sequence that is required for target cleavage, or it may be some combination of nucleotides from both strands. In the case of the CasX proteins disclosed herein, the PAM is located 5′ of the protospacer with a single nucleotide separating the PAM from the first nucleotide of the protospacer. Thus, in the case of reference CasX, a TTC PAM should be understood to mean a sequence following the formula 5′- . . . NNTTCN(protospacer)NNNNNN . . . 3′ (SEQ ID NO: 3296) where ‘N’ is any DNA nucleotide and ‘(protospacer)’ is a DNA sequence having identity with the targeting sequence of the guide RNA. In the case of a CasX variant with expanded PAM recognition, a TTC, CTC, GTC, or ATC PAM should be understood to mean a sequence following the formulae; 5′- . . . NNTTCN(protospacer)NNNNNN . . . 3′ (SEQ ID NO: 3296); 5′- . . . NNCTCN(protospacer)NNNNNN . . . 3′ (SEQ ID NO: 3297); 5′- . . . NNGTCN(protospacer)NNNNNN . . . 3′ (SEQ ID NO: 3298); or 5′- . . . NNATCN(protospacer)NNNNNN . . . 3′ (SEQ ID NO: 3299). Alternatively, a TC PAM should be understood to mean a sequence following the formula 5′- . . . NNNTCN(protospacer)NNNNNN . . . 3′ (SEQ ID NO: 3300).

In some embodiments, a CasX variant has improved editing of a PAM sequence exhibits greater editing efficiency and/or binding of a target sequence in the target DNA when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5′ to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a cellular assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX protein in a comparable assay system. In some embodiments, the PAM sequence is TTC. In some embodiments, the PAM sequence is ATC. In some embodiments, the PAM sequence is CTC. In some embodiments, the PAM sequence is GTC.

q. Unwinding of DNA

In some embodiments, a CasX variant protein has improved ability to unwind DNA relative to a reference CasX protein. Poor dsDNA unwinding has been shown previously to impair or prevent the ability of CRISPR/Cas system proteins AnaCas9 or Cas14s to cleave DNA. Therefore, without wishing to be bound by any theory, it is likely that increased DNA cleavage activity by some CasX variant proteins of the disclosure is due, at least in part, to an increased ability to find and unwind the dsDNA at a target site. Methods of measuring the ability of CasX proteins (such as variant or reference) to unwind DNA include, but are not limited to, in vitro assays that observe increased on rates of dsDNA targets in fluorescence polarization or biolayer interferometry.

Without wishing to be bound by theory, it is thought that amino acid changes in the NTSB domain may produce CasX variant proteins with increased DNA unwinding characteristics. Alternatively, or in addition, amino acid changes in the OBD or the helical domain regions that interact with the PAM may also produce CasX variant proteins with increased DNA unwinding characteristics.

r. Catalytic Activity

The ribonucleoprotein complex of the CasX:gNA systems disclosed herein comprise a reference CasX protein or CasX variant complexed with a gNA that binds to a target nucleic acid and, in some cases, cleaves the target nucleic acid. In some embodiments, a CasX variant protein has improved catalytic activity relative to a reference CasX protein. Without wishing to be bound by theory, it is thought that in some cases cleavage of the target strand can be a limiting factor for Cas12-like molecules in creating a dsDNA break. In some embodiments, CasX variant proteins improve bending of the target strand of DNA and cleavage of this strand, resulting in an improvement in the overall efficiency of dsDNA cleavage by the CasX ribonucleoprotein complex.

In some embodiments, a CasX variant protein has increased nuclease activity compared to a reference CasX protein. Variants with increased nuclease activity can be generated, for example, through amino acid changes in the RuvC nuclease domain. In some embodiments, amino acid substitutions in amino acid residues 708-804 of the RuvC domain can result in increased editing efficiency, as seen in FIG. 10. In some embodiments, the CasX variant comprises a nuclease domain having nickase activity. In the foregoing embodiment, the CasX nickase of a gene editing pair generates a single-stranded break within 10-18 nucleotides 3′ of a PAM site in the non-target strand. In other embodiments, the CasX variant comprises a nuclease domain having double-stranded cleavage activity. In the foregoing, the CasX of the gene editing pair generates a double-stranded break within 18-26 nucleotides 5′ of a PAM site on the target strand and 10-18 nucleotides 3′ on the non-target strand. Nuclease activity can be assayed by a variety of methods, including those of the Examples. In some embodiments, a CasX variant has a K_(cleave) constant that is at least 2-fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold, or at least 9-fold, or at least 10-fold greater compared to a reference or wild-type CasX.

In some embodiments, a CasX variant protein has increased target strand loading for double strand cleavage. Variants with increased target strand loading activity can be generated, for example, through amino acid changes in the TLS domain. Without wishing to be bound by theory, amino acid changes in the TSL domain may result in CasX variant proteins with improved catalytic activity. Alternatively, or in addition, amino acid changes around the binding channel for the RNA:DNA duplex may also improve catalytic activity of the CasX variant protein.

In some embodiments, a CasX variant protein has increased collateral cleavage activity compared to a reference CasX protein. As used herein, “collateral cleavage activity” refers to additional, non-targeted cleavage of nucleic acids following recognition and cleavage of a target nucleic acid. In some embodiments, a CasX variant protein has decreased collateral cleavage activity compared to a reference CasX protein.

In some embodiments, for example those embodiments encompassing applications where cleavage of the target nucleic acid is not a desired outcome, improving the catalytic activity of a CasX variant protein comprises altering, reducing, or abolishing the catalytic activity of the CasX variant protein. In some embodiments, a ribonucleoprotein complex comprising a dCasX variant protein binds to a target nucleic acid and does not cleave the target nucleic acid.

In some embodiments, the CasX ribonucleoprotein complex comprising a CasX variant protein binds a target DNA but generates a single stranded nick in the target DNA. In some embodiments, particularly those embodiments wherein the CasX protein is a nickase, a CasX variant protein has decreased target strand loading for single strand nicking. Variants with decreased target strand loading may be generated, for example, through amino acid changes in the TSL domain.

Exemplary methods for characterizing the catalytic activity of CasX proteins may include, but are not limited to, in vitro cleavage assays, including those of the Examples, below. In some embodiments, electrophoresis of DNA products on agarose gels can interrogate the kinetics of strand cleavage.

s. Affinity for Target RNA

In some embodiments, a ribonucleoprotein complex comprising a reference CasX protein or variant thereof binds to a target RNA and cleaves the target nucleic acid. In some embodiments, variants of a reference CasX protein increase the specificity of the CasX variant protein for a target RNA, and increase the activity of the CasX variant protein with respect to a target RNA when compared to the reference CasX protein. For example, CasX variant proteins can display increased binding affinity for target RNAs, or increased cleavage of target RNAs, when compared to reference CasX proteins. In some embodiments, a ribonucleoprotein complex comprising a CasX variant protein binds to a target RNA and/or cleaves the target RNA. In some embodiments, a CasX variant has at least about two-fold to about 10-fold increased binding affinity to the target nucleic acid compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

t. CasX Fusion Proteins

In some embodiments, the disclosure provides CasX proteins comprising a heterologous protein fused to the CasX. In some cases, the CasX is a reference CasX protein. In other cases, the CasX is a CasX variant of any of the embodiments described herein.

In some embodiments, the CasX variant protein is fused to one or more proteins or domains thereof that have a different activity of interest, resulting in a fusion protein. For example, in some embodiments, the CasX variant protein is fused to a protein (or domain thereof) that inhibits transcription, modifies a target nucleic acid, or modifies a polypeptide associated with a nucleic acid (e.g., histone modification).

In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 fused to one or more proteins or domains thereof with an activity of interest. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 fused to one or more proteins or domains thereof with an activity of interest. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 fused to one or more proteins or domains thereof with an activity of interest.

In some embodiments, a heterologous polypeptide (or heterologous amino acid such as a cysteine residue or a non-natural amino acid) can be inserted at one or more positions within a CasX protein to generate a CasX fusion protein. In other embodiments, a cysteine residue can be inserted at one or more positions within a CasX protein followed by conjugation of a heterologous polypeptide described below. In some alternative embodiments, a heterologous polypeptide or heterologous amino acid can be added at the N- or C-terminus of the reference or CasX variant protein. In other embodiments, a heterologous polypeptide or heterologous amino acid can be inserted internally within the sequence of the CasX protein.

In some embodiments, the reference CasX or variant fusion protein retains RNA-guided sequence specific target nucleic acid binding and cleavage activity. In some cases, the reference CasX or variant fusion protein has (retains) 50% or more of the activity (e.g., cleavage and/or binding activity) of the corresponding reference CasX or variant protein that does not have the insertion of the heterologous protein. In some cases, the reference CasX or variant fusion protein retains at least about 60%, or at least about 70%, at least about 80%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or about 100% of the activity (e.g., cleavage and/or binding activity) of the corresponding CasX protein that does not have the insertion of the heterologous protein.

In some cases, the reference CasX or CasX variant fusion protein retains (has) target nucleic acid binding activity relative to the activity of the CasX protein without the inserted heterologous amino acid or heterologous polypeptide. In some cases, the reference CasX or CasX variant fusion protein retains at least about 60%, or at least about 70%, at least about 80%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or about 100% of the binding activity of the corresponding CasX protein that does not have the insertion of the heterologous protein.

In some cases, the reference CasX or CasX variant fusion protein retains (has) target nucleic acid binding and/or cleavage activity relative to the activity of the parent CasX protein without the inserted heterologous amino acid or heterologous polypeptide. For example, in some cases, the reference CasX or CasX variant fusion protein has (retains) 50% or more of the binding and/or cleavage activity of the corresponding parent CasX protein (the CasX protein that does not have the insertion). For example, in some cases, the reference CasX or CasX variant fusion protein has (retains) 60% or more (70% or more, 80% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 100%) of the binding and/or cleavage activity of the corresponding CasX parent protein (the CasX protein that does not have the insertion). Methods of measuring cleaving and/or binding activity of a CasX protein and/or a CasX fusion protein will be known to one of ordinary skill in the art, and any convenient method can be used.

A variety of heterologous polypeptides are suitable for inclusion in a reference CasX or CasX variant fusion protein of the disclosure. In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like). In some cases the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like).

In some cases, a fusion partner has enzymatic activity that modifies a target nucleic acid; e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity.

In some cases, a fusion partner has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid; e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a polypeptide with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a polypeptide with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a polypeptide with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity.

Examples of proteins (or fragments thereof) that can be used as a suitable fusion partner to a reference CasX or CasX variant to increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or transcription activator-like (TAL) activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET domain containing 1A, histone lysine methyltransferase (SET1A), SET domain containing 1B, histone lysine methyltransferase (SET1B), lysine methyltransferase 2A (MLL1) to 5, ASCL1 (ASH1) achaete-scute family bHLH transcription factor 1 (ASH1), SET and MYND domain containing 2 provided (SMYD2), nuclear receptor binding SET domain protein 1 (NSD1), and the like; histone lysine demethylases such as lysine demethylase 3A (JHDM2a)/Lysine-specific demethylase 3B (JHDM2b), lysine demethylase 6A (UTX), lysine demethylase 6B (JMJD3), and the like; histone acetyltransferases such as lysine acetyltransferase 2A (GCN5), lysine acetyltransferase 2B (PCAF), CREB binding protein (CBP), E1A binding protein p30 (p300), TATA-box binding protein associated factor 1 (TAF1), lysine acetyltransferase 5 (TIP60/PLIP), lysine acetyltransferase 6A (MOZ/MYST3), lysine acetyltransferase 6B (MORF/MYST4), SRC proto-oncogene, non-receptor tyrosine kinase (SRC1), nuclear receptor coactivator 3 (ACTR), MYB binding protein 1a (P160), clock circadian regulator (CLOCK), and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), tet methylcytosine dioxygenase 1 (TET1), demeter (DME), demeter-like 1 (DML1), demeter-like 2 (DML2), protein ROS1 (ROS1), and the like.

Examples of proteins (or fragments thereof) that can be used as a suitable fusion partner with a reference CasX or CasX variant to decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as PR/SET domain containing protein (Pr-SET)7/8, lysine methyltransferase 5B (SUV4-20H1), PR/SET domain 2 (RIZ1), and the like; histone lysine demethylases such as lysine demethylase 4A (JMJD2A/JHDM3A), lysine demethylase 4B (JMJD2B), lysine demethylase 4C (JMJD2C/GASC1), lysine demethylase 4D (JMJD2D), lysine demethylase 5A (JARID1A/RBP2), lysine demethylase 5B (JARID1B/PLU-1), lysine demethylase 5C (JARID 1C/SMCX), lysine demethylase 5D (JARID1D/SMCY), and the like; histone lysine deacetylases such as histone deacetylase 1 (HDAC1), HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, sirtuin 1 (SIRT1), SIRT2, HDAC11, and the like; DNA methylases such as HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), methyltransferase 1 (MET1), S-adenosyl-L-methionine-dependent methyltransferases superfamily protein (DRM3) (plants), DNA cytosine methyltransferase MET2a (ZMET2), chromomethylase 1 (CMT1), chromomethylase 2 (CMT2) (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

In some cases, the fusion partner to a reference CasX or CasX variant has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET 1 CD), TET1, DME, DML1, DML2, ROS1, and the like), DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme, e.g., an APOBEC protein such as rat apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 {APOBEC1}), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).

In some cases, a reference CasX or CasX variant protein of the present disclosure is fused to a polypeptide selected from: a domain for increasing transcription (e.g., a VP16 domain, a VP64 domain), a domain for decreasing transcription (e.g., a KRAB domain. e.g., from the Kox1 protein), a core catalytic domain of a histone acetyltransferase (e.g., histone acetyltransferase p300), a protein/domain that provides a detectable signal (e.g., a fluorescent protein such as GFP), a nuclease domain (e.g., a Fokl nuclease), and a base editor (discussed further below).

In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 fused to a polypeptide selected from the group consisting of a domain for decreasing transcription, a domain with enzymatic activity, a core catalytic domain of a histone acetyltransferase, a protein/domain that provides a detectable signal, a nuclease domain, and a base editor. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 fused to a polypeptide selected from the group consisting of a domain for decreasing transcription, a domain with enzymatic activity, a core catalytic domain of a histone acetyltransferase, a protein/domain that provides a detectable signal, a nuclease domain, and a base editor. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 fused to a polypeptide selected from the group consisting of a domain for decreasing transcription, a domain with enzymatic activity, a core catalytic domain of a histone acetyltransferase, a protein/domain that provides a detectable signal, a nuclease domain, and a base editor.

In some cases, a reference CasX protein or CasX variant of the present disclosure is fused to a base editor. Base editors include those that can alter a guanine, adenine, cytosine, thymine, or uracil base on a nucleoside or nucleotide. Base editors include, but are not limited to an adenosine deaminase, cytosine deaminase (e.g. APOBEC1), and guanine oxidase. Accordingly, any of the reference CasX or CasX variants provided herein may comprise (i.e., are fused to) a base editor; for example a reference CasX or CasX variant of the disclosure may be fused to an adenosine deaminase, a cytosine deaminase, or a guanine oxidase. In exemplary embodiments, a CasX variant of the disclosure comprising any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 is fused to an adenosine deaminase, cytosine deaminase, or a guanine oxidase.

In some cases, the fusion partner to a reference CasX or CasX variant has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the fusion partner with a reference CasX or CasX variant include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB 1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SMYD2, NSD1, DOT1 like histone lysine methyltransferase (DOT1L), Pr-SET7/8, lysine methyltransferase 5B (SUV4-20H1), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), PR/SET domain 2 (RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HB01/MYST2, HMOF/MYST1, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity. SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

Additional examples of suitable fusion partners to a reference CasX or CasX variant are (i) a dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable subject RNA-guided polypeptide), and (ii) a chloroplast transit peptide.

Suitable chloroplast transit peptides include, but are not limited to sequences having at least 80%, at least 90%, or at least 95% identity to or are identical to: MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGR VKCMQVWPPIGKKKFETLSYLPPLTRDSRA (SEQ ID NO: 338); MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSTTSNGGRVKS (SEQ ID NO: 339); MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQV WPPIEKKKFETLSYLPDLTDSGGRVNC (SEQ ID NO: 340); MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG SELRPLKVMSSVSTAC (SEQ ID NO: 341); MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG SELRPLKVMSSVSTAC (SEQ ID NO: 342); MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLKKDSIFMQLF CSFRISASVATAC (SEQ ID NO: 343); MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASAAPKQSRKPH RFDRRCLSMVV (SEQ ID NO: 344); MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQ QRSVQRGSRRFPSVVVC (SEQ ID NO: 345); MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC (SEQ ID NO: 346); MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAAVTPQASPVIS RSAAAA (SEQ ID NO: 347); and MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTING AAATTNGASAASS (SEQ ID NO: 348). In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a chloroplast transit peptide. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a chloroplast transit peptide. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a chloroplast transit peptide.

In some cases, a reference CasX or CasX variant protein of the present disclosure can include an endosomal escape peptide. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 349), wherein each X is independently selected from lysine, histidine, and arginine. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 350), or HHHHHHHHH (SEQ ID NO: 351). In some embodiments, a CasX variant comprises a sequence of any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and an endosomal escape polypeptide. In some embodiments, a CasX variant comprises a sequence of any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and an endosomal escape polypeptide. In some embodiments, a CasX variant comprises a sequence of any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and an endosomal escape polypeptide.

Non-limiting examples of suitable fusion partners for a reference CasX or CasX variant for use when targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eukaryotic translation initiation factor 4 gamma {eIF4G}); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain). In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a protein or domain selected from the group consisting of a splicing factor, a protein translation component, an RNA methylase, an RNA editing enzyme, a helicase, and an RNA binding protein. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a protein or domain selected from the group consisting of a splicing factor, a protein translation component, an RNA methylase, an RNA editing enzyme, a helicase, and an RNA binding protein. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a protein or domain selected from the group consisting of a splicing factor, a protein translation component, an RNA methylase, an RNA editing enzyme, a helicase, and an RNA binding protein.

A fusion partner for a reference CasX or CasX variant can be any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and/or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising; endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example cleavage and polyadenylation specific factor {CPSF}, cleavage stimulation factor {CstF}, CFIm and CFIIm); exonucleases (for example chromatin-binding exonuclease XRN1 (XRN-1) or Exonuclease T); deadenylases (for example DNA 5′-adenosine monophosphate hydrolase {HNT3}); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1 RNA helicase and ATPase {UPF1}, UPF2, UPF3, UPF3b, RNP SI, RNA binding motif protein 8A {Y14}, DEK proto-oncogene {DEK}, RNA-processing protein REF2 {REF2}, and Serine-arginine repetitive matrix 1 {SRm160}); proteins and protein domains responsible for stabilizing RNA (for example poly(A) binding protein cytoplasmic 1 {PABP}); proteins and protein domains responsible for repressing translation (for example argonaute RISC catalytic component 2 {Ago2} and Ago4); proteins and protein domains responsible for stimulating translation (for example Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (for example poly(A) polymerase (PAP1), PAP-associated domain-containing protein; Poly(A) RNA polymerase gld-2 {GLD-2}, and Star-PAP); proteins and protein domains responsible for polyuridinylation of RNA (for example Terminal uridylyltransferase {CID1} and terminal uridylate transferase); proteins and protein domains responsible for RNA localization (for example from insulin like growth factor 2 mRNA binding protein 1 {IMP1}, Z-DNA binding protein 1 {ZBP1}, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (for example Rrp6); proteins and protein domains responsible for nuclear export of RNA (for example nuclear RNA export factor 1 {TAP}, nuclear RNA export factor 1 {NXF1}, THO Complex {THO}, TREX, REF, and Aly/REF export factor {Aly}); proteins and protein domains responsible for repression of RNA splicing (for example polypyrimidine tract binding protein 1 {PTB}, KH RNA binding domain containing, signal transduction associated 1 Sam68), and heterogeneous nuclear ribonucleoprotein A1 {hnRNP A1}); proteins and protein domains responsible for stimulation of RNA splicing (for example serine/arginine-rich (SR) domains); proteins and protein domains responsible for reducing the efficiency of transcription (for example FUS RNA binding protein {FUS (TLS)}); and proteins and protein domains responsible for stimulating transcription (for example cyclin dependent kinase 7 {CDK7} and HIV Tat). Alternatively, the effector domain may be selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having nonsense mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in more detail in WO2012068627, which is hereby incorporated by reference in its entirety.

Some suitable RNA splicing factors that can be used (in whole or as fragments thereof) as a fusion partner with a reference CasX or CasX variant have modular organization, with separate sequence-specific RNA binding modules and splicing effector domains. For example, members of the serine/arginine-rich (SR) protein family contain N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal glycine-rich domain. Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF/SF2 can recognize ESEs and promote the use of intron proximal sites, whereas hnRNP A1 can bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, BCL2 like 1 (Bcl-x) pre-mRNA produces two splicing isoforms with two alternative 5′ splice sites to encode proteins of opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived post mitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple cc-elements that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5′ splice sites). For more examples, see WO2010075303, which is hereby incorporated by reference in its entirety. Further suitable fusion partners include, but are not limited to proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.).

In some cases, a heterologous polypeptide (a fusion partner) for use with a reference CasX or CasX variant provides for subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like). In some embodiments, a subject RNA-guided polypeptide or a conditionally active RNA-guided polypeptide and/or subject CasX fusion protein does not include a NLS so that the protein is not targeted to the nucleus, which can be advantageous; e.g., when the target nucleic acid is an RNA that is present in the cytosol. In some embodiments, a fusion partner can provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g., a 6×His tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like). In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a subcellular localization sequence or a tag. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a subcellular localization sequence or a tag. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a subcellular localization sequence or a tag.

In some cases, a reference or CasX variant protein includes (is fused to) a nuclear localization signal (NLS). In some cases, a reference or CasX variant protein is fused to 2 or more, 3 or more, 4 or more, or 5 or more 6 or more, 7 or more, 8 or more NLSs. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus. In some cases, a reference or CasX variant protein includes (is fused to) between 1 and 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NLSs). In some cases, a reference or CasX variant protein includes (is fused to) between 2 and 5 NLSs (e.g., 2-4, or 2-3 NLSs).

Non-limiting examples of NLSs suitable for use with a reference CasX or CasX variant include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to sequences derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 352); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 353); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 354) or RQRRNELKRSP (SEQ ID NO: 355); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 356); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 357) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 358) and PPKKARED (SEQ ID NO: 359) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 360) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 361) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 362) and PKQKKRK (SEQ ID NO: 363) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 364) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 365) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 366) of the human poly(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 367) of the steroid hormone receptors (human) glucocorticoid; the sequence PRPRKIPR (SEQ ID NO: 368) of Borna disease virus P protein (BDV-P1); the sequence PPRKKRTVV (SEQ ID NO: 369) of hepatitis C virus nonstructural protein (HCV-NS5A); the sequence NLSKKKKRKREK (SEQ ID NO: 370) of LEF1; the sequence RRPSRPFRKP (SEQ ID NO: 371) of ORF57 simirae; the sequence KRPRSPSS (SEQ ID NO: 372) of EBV LANA; the sequence KRGINDRNFWRGENERKTR (SEQ ID NO: 373) of Influenza A protein; the sequence PRPPKMARYDN (SEQ ID NO: 374) of human RNA helicase A (RHA); the sequence KRSFSKAF (SEQ ID NO: 375) of nucleolar RNA helicase II; the sequence KLKIKRPVK (SEQ ID NO: 376) of TUS-protein; the sequence PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 377) associated with importin-alpha; the sequence PKTRRRPRRSQRKRPPT (SEQ ID NO: 378) from the Rex protein in HTLV-1; the sequence SRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 379) from the EGL-13 protein of Caenorhabditis elegans; and the sequences KTRRRPRRSQRKRPPT (SEQ ID NO: 380), RRKKRRPRRKKRR (SEQ ID NO: 381), PKKKSRKPKKKSRK (SEQ ID NO: 382), HKKKHPDASVNFSEFSK (SEQ ID NO: 383), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 384), LSPSLSPLLSPSLSPL (SEQ ID NO: 385), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 386), PKRGRGRPKRGRGR (SEQ ID NO: 387), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 388) and PKKKRKVPPPPKKKRKV (SEQ ID NO: 389). In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of a reference or CasX variant fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to a reference or CasX variant fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.

In some embodiments, a CasX variant comprising an N terminal NLS comprises a sequence of any one of SEQ ID NOS: 3508-3540-3549. In some embodiments, a CasX variant comprising an N terminal NLS comprises a sequence with one or more additional modifications to of any one of SEQ ID NOS: 3508-3540-3549.

In some cases, a reference or CasX variant fusion protein includes a “Protein Transduction Domain” or PTD (also known as a CPP—cell penetrating peptide), which refers to a protein, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from an extracellular space to an intracellular space, or from the cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino terminus of a reference or CasX variant fusion protein. In some embodiments, a PTD is covalently linked to the carboxyl terminus of a reference or CasX variant fusion protein. In some cases, the PTD is inserted internally in the sequence of a reference or CasX variant fusion protein at a suitable insertion site. In some cases, a reference or CasX variant fusion protein includes (is conjugated to, is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, a PTD includes one or more nuclear localization signals (NLS). Examples of PTDs include but are not limited to peptide transduction domain of HIV TAT comprising YGRKKRRQRRR (SEQ ID NO: 390), RKKRRQRR (SEQ ID NO: 391); YARAAARQARA (SEQ ID NO: 392); THRLPRRRRRR (SEQ ID NO: 393); and GGRRARRRRRR (SEQ ID NO: 394); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO: 395); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 396); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 397); and RQIKIWFQNRRMKWKK (SEQ ID NO: 398). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a PTD. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a PTD. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a PTD.

In some embodiments, a reference or CasX variant fusion protein can include a CasX protein that is linked to an internally inserted heterologous amino acid or heterologous polypeptide (a heterologous amino acid sequence) via a linker polypeptide (e.g., one or more linker polypeptides). In some embodiments, a reference or CasX variant fusion protein can be linked at the C-terminal and/or N-terminal end to a heterologous polypeptide (fusion partner) via a linker polypeptide (e.g., one or more linker polypeptides) The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers are generally produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. Example linker polypeptides include glycine polymers (G)n, glycine-serine polymer (including, for example, (GS)n, GSGGSn (SEQ ID NO: 399), GGSGGSn (SEQ ID NO: 400), and GGGSn (SEQ ID NO: 401), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, glycine-proline polymers, proline polymers and proline-alanine polymers. Example linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 402), GGSGG (SEQ ID NO: 403), GSGSG (SEQ ID NO: 404), GSGGG (SEQ ID NO: 405), GGGSG (SEQ ID NO: 406), GSSSG (SEQ ID NO: 407), GPGP (SEQ ID NO: 408), GGP, PPP, PPAPPA (SEQ ID NO: 409), PPPGPPP (SEQ ID NO: 410) and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.

V. gNA and CasX Protein Gene Editing Pairs

In other aspects, provided herein are compositions of a gene editing pair comprising a CasX protein and a guide NA, referred to herein as a gene editing pair. In certain embodiments, the gene editing pair comprises a CasX variant protein as described herein (e.g., any one of the sequences set forth in Tables 3, 8, 9, 10 and 12) or a reference CasX protein as described herein (e.g., SEQ ID NOS:1-3), while, the guide NA is a reference gRNA (SEQ ID NOS: 4-16) or a gNA variant as described herein (e.g., SEQ ID NOS: 2101-2280), or sequence variants having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity thereto, wherein the gNA comprises a targeting sequence complementary to the target DNA. In those embodiments in which one component is a variant, the pair is referred to as a variant gene editing pair. In other embodiments, a gene editing pair comprises the CasX protein, a first gNA (either a reference gRNA {SEQ ID NOS: 4-16} or a gNA variant as described herein {e.g., SEQ ID NOS: 2101-2280}) with a targeting sequence, and a second gNA variant or a second reference guide nucleic acid, wherein the second gNA variant or the second reference guide nucleic acid has a targeting sequence complementary to a different or overlapping portion of the target DNA compared to the targeting sequence of the first gNA.

In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair, wherein the reference gene editing pair comprises a CasX protein of SEQ ID NOS: 1-3, a different gNA, or both. For example, in some embodiments, the variant gene editing pair comprises a CasX variant protein, and the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein. In other embodiments, the variant gene editing pair comprises a gNA variant, and the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference gRNA. In other embodiments, the variant gene editing pair comprises a gNA variant and a CasX variant protein, and the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein and a reference gRNA.

In some embodiments of the variant gene editing pairs provided herein, the CasX is a variant protein as described herein (e.g., the sequences set forth in Tables 3, 8, 9, 10 and 12 or sequence variants having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to the listed sequences) while the gNA is a reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4. In some embodiments of the variant gene editing pairs provided herein, the CasX comprises a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 while the gNA variant is a sequence of SEQ ID NOS:2101-2280, or sequence variants having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity to the listed sequences.

In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4. In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and a reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4.

Exemplary improved characteristics, as described herein, may in some embodiments, and include improved CasX:gNA RNP complex stability, improved binding affinity between the CasX and gNA, improved kinetics of RNP complex formation, higher percentage of cleavage-competent RNP, improved RNP binding affinity to the target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity. In the foregoing embodiments, the improvement is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the characteristic of a reference CasX protein and reference gNA pair. In other cases, the one or more of the improved characteristics may be improved about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to a reference gene editing pair. In other cases, the one or more of the improved characteristics may be improved about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold or more improved relative to a reference gene editing pair.

In some embodiments, the variant gene editing pair comprises a gNA variant comprising a sequence of any one of SEQ ID NOs: 2101-2280 and a reference CasX protein comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant gene editing pair comprises a gNA variant comprising a sequence of any one of SEQ ID NOS: 2101-2280 and a CasX variant protein comprising a variant of the reference CasX protein of SEQ ID NO: 2. In some embodiments, the variant gene editing pair comprises a reference gRNA comprising a sequence of SEQ ID NO: 5 or SEQ ID NO: 4 and a CasX variant protein comprising a variant of the reference CasX protein of SEQ ID NO: 2. In some embodiments, the CasX variant protein comprises a Y789T substitution of SEQ ID NO: 2; a deletion of P at position 793 of SEQ ID NO: 2, a Y789D substitution of SEQ ID NO: 2, a T72S substitution of SEQ ID NO: 2, a I546V substitution of SEQ ID NO: 2, a E552A substitution of SEQ ID NO: 2, a A636D substitution of SEQ ID NO: 2, a F536S substitution of SEQ ID NO: 2, a A708K substitution of SEQ ID NO: 2, a Y797L substitution of SEQ ID NO: 2, a L792G substitution of SEQ ID NO: 2, a A739V substitution of SEQ ID NO: 2, a G791M substitution of SEQ ID NO: 2, an insertion of A at position 661 of SEQ ID NO: 2, a A788W substitution of SEQ ID NO: 2, a K390R substitution of SEQ ID NO: 2, a A751 S substitution of SEQ ID NO: 2, a E385A substitution of SEQ ID NO: 2, a combination of S794R and Y797L substitutions of SEQ ID NO: 2, an insertion of P at 696 of SEQ ID NO: 2, a combination of K416E and A708K substitutions of SEQ ID NO: 2, an insertion of M at position 773 of SEQ ID NO: 2, a G695H substitution of SEQ ID NO: 2, an insertion of AS at position 793 of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, a C477R substitution of SEQ ID NO: 2, a C477K substitution of SEQ ID NO: 2, a C479A substitution of SEQ ID NO: 2, a C479L substitution of SEQ ID NO: 2, a combination of an A708K substitution and a deletion of P at position 793 of SEQ ID NO: 2, a I55F substitution of SEQ ID NO: 2, a K210R substitution of SEQ ID NO: 2, a C233S substitution of SEQ ID NO: 2, a D231N substitution of SEQ ID NO: 2, a Q338E substitution of SEQ ID NO: 2, a Q338R substitution of SEQ ID NO: 2, a L379R substitution of SEQ ID NO: 2, a K390R substitution of SEQ ID NO: 2, a L481Q substitution of SEQ ID NO: 2, a F495S substitution of SEQ ID NO: 2, a D600N substitution of SEQ ID NO: 2, a T886K substitution of SEQ ID NO: 2, a combination of a deletion of P at position 793J and a P793AS substitution of SEQ ID NO: 2, a A739V substitution of SEQ ID NO: 2, a K460N substitution of SEQ ID NO: 2, a I199F substitution of SEQ ID NO: 2, a G492P substitution of SEQ ID NO: 2, a T153I substitution of SEQ ID NO: 2, a R591I substitution of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, an insertion of AS at position 796 of SEQ ID NO: 2, an insertion of L at position 889 of SEQ ID NO: 2, a E121D substitution of SEQ ID NO: 2, a S270W substitution of SEQ ID NO: 2, a E712Q substitution of SEQ ID NO: 2, a K942Q substitution of SEQ ID NO: 2, a E552K substitution of SEQ ID NO: 2, a K25Q substitution of SEQ ID NO: 2, a N47D substitution of SEQ ID NO: 2, a combination Q367K and I425S substitutions of SEQ ID NO: 2, an insertion of T at position 696 of SEQ ID NO: 2, a L685I substitution of SEQ ID NO: 2, a N880D substitution of SEQ ID NO: 2, a combination of a A708K substitution, a deletion of P at position 793 and a A739V substitution of SEQ ID NO: 2, a Q102R substitution of SEQ ID NO: 2, a M734K substitution of SEQ ID NO: 2, a A724S substitution of SEQ ID NO: 2, a T704K substitution of SEQ ID NO: 2, a P224K substitution of SEQ ID NO: 2, a combination of Q338R and A339E substitutions of SEQ ID NO: 2, a combination of Q338R and A339K substitutions of SEQ ID NO: 2, a K25R substitution of SEQ ID NO: 2, a M29E substitution of SEQ ID NO: 2, a H152D substitution of SEQ ID NO: 2, a S219R substitution of SEQ ID NO: 2, a E475K substitution of SEQ ID NO: 2, a combination of S507G and G508R substitutions of SEQ ID NO: 2, a g226R substitution of SEQ ID NO: 2, a A377K substitution of SEQ ID NO: 2, a E480K substitution of SEQ ID NO: 2, a K416E substitution of SEQ ID NO: 2, a H164R substitution of SEQ ID NO: 2, a K767R substitution of SEQ ID NO: 2, a I7F substitution of SEQ ID NO: 2, a m29R substitution of SEQ ID NO: 2, a H435R substitution of SEQ ID NO: 2, a E385Q substitution of SEQ ID NO: 2, a E385K substitution of SEQ ID NO: 2, a I279F substitution of SEQ ID NO: 2, a D489S substitution of SEQ ID NO: 2, a D732N substitution of SEQ ID NO: 2, a A739T substitution of SEQ ID NO: 2, a W885R substitution of SEQ ID NO: 2, a E53K substitution of SEQ ID NO: 2, a A238T substitution of SEQ ID NO: 2, a P283Q substitution of SEQ ID NO: 2, a E292K substitution of SEQ ID NO: 2, a Q628E substitution of SEQ ID NO: 2, a combination of F5561+D646A+G695D+A751S+A820P substitutions of SEQ ID NO: 2, a R388Q substitution of SEQ ID NO: 2, a combination of L491I and M771N substitutions of SEQ ID NO: 2, a G791M substitution of SEQ ID NO: 2, a L792K substitution of SEQ ID NO: 2, a L792E substitution of SEQ ID NO: 2, a M779N substitution of SEQ ID NO: 2, a G27D substitution of SEQ ID NO: 2, a combination of L379R and A708K substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of C477K and A708K substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of L379R, C477K and A708K substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of L379R, A708K and A739V substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of C477K, A708K and A739V substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of L379R, C477K, A708K and A739V substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a K955R substitution of SEQ ID NO: 2, a S867R substitution of SEQ ID NO: 2, a R693I substitution of SEQ ID NO: 2, a F189Y substitution of SEQ ID NO: 2, a V635M substitution of SEQ ID NO: 2, a F399L substitution of SEQ ID NO: 2, a E498K substitution of SEQ ID NO: 2, a E386R substitution of SEQ ID NO: 2, a V254G substitution of SEQ ID NO: 2, a P793S substitution of SEQ ID NO: 2, a K188E substitution of SEQ ID NO: 2, a QT945KI substitution of SEQ ID NO: 2, a T620P substitution of SEQ ID NO: 2, a T946P substitution of SEQ ID NO: 2, a TI49PP substitution of SEQ ID NO: 2, a N952T substitution of SEQ ID NO: 2 or a K682E substitution of SEQ ID NO: 2.

In some embodiments, the variant gene editing pair comprises a CasX gRNA of SEQ ID NO: 5 and a CasX variant protein comprising a combination of L379R and A708K substitutions and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, the variant gene editing pair comprises a reference CasX protein SEQ ID NO: 2 and sgNA scaffold variant of SEQ ID NO: 5.

In some embodiments of the sgNA:protein variant pairs of the disclosure, the CasX variant protein is selected from the group consisting of: a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of T620P of SEQ ID NO: 2; a CasX variant protein comprising a substitution of M771A of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of W782Q of SEQ ID NO: 2; a CasX variant protein comprising a substitution of M771Q of SEQ ID NO: 2; a CasX variant protein comprises a substitution of R458I and a substitution of A739V of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D489S of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of V711K of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of A708K, a substitution of P at position 793 and a substitution of E386S of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L792D of SEQ ID NO: 2; a CasX variant protein comprising a substitution of G791F of SEQ ID NO: 2; a CasX variant protein comprising a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2; a CasX variant protein comprising a substitution of C477K, a substitution of A708K and a substitution of P at position 793 of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L2491 and a substitution of M771N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of V747K of SEQ ID NO: 2; and a CasX variant protein comprises a substitution of L379R, a substitution of C477, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO: 2; and the sequence encoding the sgNA variant is selected from the group consisting of SEQ ID NO: 2104, SEQ ID NO: 2163, SEQ ID NO: 2107, SEQ ID NO: 2164, SEQ ID NO: 2165, SEQ ID NO: 2166, SEQ ID NO: 2103, SEQ ID NO: 2167, SEQ ID NO: 2105, SEQ ID NO: 2108, SEQ ID NO: 2112, SEQ ID NO: 2160, SEQ ID NO: 2170, SEQ ID NO: 2114, SEQ ID NO: 2171, SEQ ID NO: 2112, SEQ ID NO: 2173, SEQ ID NO: 2102, SEQ ID NO: 2174, SEQ ID NO: 2175, SEQ ID NO: 2109, SEQ ID NO: 2176, SEQ ID NO: 2238, or SEQ ID NO: 2239.

In some embodiments, the gene editing pair comprises a CasX selected from any one of CasX of sequence SEQ ID NO: 270, SEQ ID NO: 292, SEQ ID NO: 311, SEQ ID NO: 333, or SEQ ID NO: 336, and a gNA selected from any one of SEQ ID NOS: 2104, 2106, or 2238.

In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549.

In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a gNA selected from the group consisting of any one of SEQ ID NOS: 412-3295. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, and a gNA selected from the group consisting of any one of SEQ ID NOS: 412-3295. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549, and a gNA selected from the group consisting of any one of SEQ ID NOS: 412-3295.

In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a gNA selected from the group consisting of any one of SEQ ID NOS: 2101-2280. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, and a gNA selected from the group consisting of any one of SEQ ID NOS: 2101-2280. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549, and a gNA selected from the group consisting of any one of SEQ ID NOS: 2101-2280.

In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a gNA selected from the group consisting of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, and a gNA selected from the group consisting of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549, and a gNA selected from the group consisting of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280.

In still further embodiments, the present disclosure provides a gene editing pair comprising a CasX protein and a gNA, wherein the gNA is a guide RNA variant as described herein. In some embodiments of the gene editing pairs of the disclosure, the Cas protein is a CasX variant as described herein. In some embodiments, the CasX protein is a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the gNA is a guide RNA variant as described herein. Exemplary improved characteristics of the gene editing pair embodiments, as described herein, may in some embodiments include improved protein:gNA complex stability, improved ribonuclear protein complex (RNP) formation, higher percentage of cleavage-competent RNP, improved binding affinity between the CasX protein and gNA, improved binding affinity to the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity. In the foregoing embodiments, the improvement is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the characteristic of a reference CasX protein and reference gNA pair.

In some embodiments, wherein the gene editing pair comprises both a CasX variant protein and a gNA variant as described herein, the one or more characteristics of the gene editing pair is improved beyond what can be achieved by varying the CasX protein or the gNA alone. In some embodiments, the CasX variant protein and the gNA variant act additively to improve one or more characteristics of the gene editing pair. In some embodiments, the CasX variant protein and the gNA variant act synergistically to improve one or more characteristics of the gene editing pair. In the foregoing embodiments, the improvement is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the characteristic of a reference CasX protein and reference gNA pair.

VI. Methods of Making CasX Variant Protein and gNA Variants

The CasX variant proteins and gNA variants as described herein may be constructed through a variety of methods. Such methods may include, for example, Deep Mutational Evolution (DME), described below and in the Examples.

a. Deep Mutational Evolution (DME)

In some embodiments, DME is used to identify CasX protein and sgNA scaffold variants with improved function. The DME method, in some embodiments, comprises building and testing a comprehensive set of mutations to a starting biomolecule to produce a library of biomolecule variants; for example, a library of CasX variant proteins or sgNA scaffold variants. DME can encompass making all possible substitutions, as well as all possible small insertions, and all possible deletions of amino acids (in the case of proteins) or nucleotides (in the case of RNA or DNA) to the starting biomolecule. A schematic illustrating DME methods is shown in FIG. 1. In some embodiments, DME comprises a subset of all such possible substitutions, insertions, and deletions. In certain embodiments of DME, one or more libraries of variants are constructed, evaluated for functional changes, and this information used to construct one or more additional libraries. Such iterative construction and evaluation of variants may lead, for example, to identification of mutational themes that lead to certain functional outcomes, such as regions of the protein or RNA that when mutated in a certain way lead to one or more improved functions. Layering of such identified mutations may then further improve function, for example through additive or synergistic interactions. DME comprises library design, library construction, and library screening. In some embodiments, multiple rounds of design, construction, and screening are undertaken.

b. Library Design

DME methods produce variants of biomolecules, which are polymers of many monomers. In some embodiments, the biomolecule comprises a protein or a ribonucleic acid (RNA) molecule, wherein the monomer units are amino acids or ribonucleotides, respectively. The fundamental units of biomolecule mutation comprise either: (1) exchanging one monomer for another monomer of different identity (substitutions); (2) inserting one or more additional monomer in the biomolecule (insertions); or (3) removing one or more monomer from the biomolecule (deletions). DME libraries comprising substitutions, insertions, and deletions, alone or in combination, to any one or more monomers within any biomolecule described herein, are considered within the scope of the invention.

In some embodiments, DME is used to build and test the comprehensive set of mutations to a biomolecule, encompassing all possible substitutions, as well as small insertions and deletions of amino acids (in the case of proteins) or nucleotides (in the case of RNA). The construction and functional readout of these mutations can be achieved with a variety of established molecular biology methods. In some embodiments, the library comprises a subset of all possible modifications to monomers. For example, in some embodiments, a library collectively represents a single modification of one monomer, for at least 10% of the total monomer locations in a biomolecule, wherein each single modification is selected from the group consisting of substitution, single insertion, and single deletion. In some embodiments, the library collectively represents the single modification of one monomer, for at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or up to 100% of the total monomer locations in a starting biomolecule. In certain embodiments, for a certain percentage of the total monomer locations in a starting biomolecule, the library collectively represents each possible single modification of a one monomer, such as all possible substitutions with the 19 other naturally occurring amino acids (for a protein) or 3 other naturally occurring ribonucleotides (for RNA), insertion of each of the 20 naturally occurring amino acids (for a protein) or 4 naturally occurring ribonucleotides (for RNA), or deletion of the monomer. In still further embodiments, insertion at each location is independently greater than one monomer, for example insertion of two or more, three or more, or four or more monomers, or insertion of between one to four, between two to four, or between one to three monomers. In some embodiments, deletion at location is independently greater than one monomer, for example deletion of two or more, three or more, or four or more monomers, or deletion of between one to four, between two to four, or between one to three monomers. Examples of such libraries of CasX variants and gNA variants are described in Examples 24 and 25, respectively.

In some embodiments, the biomolecule is a protein and the individual monomers are amino acids. In those embodiments where the biomolecule is a protein, the number of possible DME mutations at each monomer (amino acid) position in the protein comprise 19 amino acid substitutions, 20 amino acid insertions and 1 amino acid deletion, leading to a total of 40 possible mutations per amino acid in the protein.

In some embodiments, a DME library of CasX variant proteins comprising insertions is 1 amino acid insertion library, a 2 amino acid insertion library, a 3 amino acid insertion library, a 4 amino acid insertion library, a 5 amino acid insertion library, a 6 amino acid insertion library, a 7 amino acid insertion library, an 8 amino acid insertion library, a 9 amino acid insertion library or a 10 amino acid insertion library. In some embodiments, a DME library of CasX variant proteins comprising insertions comprises between 1 and 4 amino acid insertions.

In some embodiments, the biomolecule is RNA. In those embodiments where the biomolecule is RNA, the number of possible DME mutations at each monomer (ribonucleotide) position in the RNA comprises 3 nucleotide substitutions, 4 nucleotide insertions, and 1 nucleotide deletion, leading to a total of 8 possible mutations per nucleotide.

In some embodiments, DME library design comprises enumerating all possible mutations for each of one or more target monomers in a biomolecule. As used herein, a “target monomer” refers to a monomer in a biomolecule polymer that is targeted for DME with the substitutions, insertions and deletions described herein. For example, a target monomer can be an amino acid at a specified position in a protein, or a nucleotide at a specified position in an RNA. A biomolecule can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more target monomers that are systematically mutated to produce a DME library of biomolecule variants. In some embodiments, every monomer in a biomolecule is a target monomer. For example, in DME of a protein where there are two target amino acids, DME library design comprises enumerating the 40 possible DME mutations at each of the two target amino acids. In a further example, in DME of an RNA where there are four target nucleotides, DME library design comprises enumerating the 8 possible DME mutations at each of the four target nucleotides. In some embodiments, each target monomer of a biomolecule is independently randomly selected or selected by intentional design. Thus, in some embodiments, a DME library comprises random variants, or variants that were designed, or variants comprising random mutations and designed mutations within a single biomolecule, or any combinations thereof.

In some embodiments of DME methods, DME mutations are incorporated into double-stranded DNA encoding the biomolecule. This DNA can be maintained and replicated in a standard cloning vector, for example a bacterial plasmid, referred to herein as the target plasmid. An exemplary target plasmid contains a DNA sequence encoding the starting biomolecule that will be subjected to DME, a bacterial origin of replication, and a suitable antibiotic resistance expression cassette. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin, ampicillin, spectinomycin, bleomycin, streptomycin, erythromycin, tetracycline or chloramphenicol. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin.

A library comprising said variants can be constructed in a variety of ways. In certain embodiments, plasmid recombineering is used to construct a library. Such methods can use DNA oligonucleotides encoding one or more mutations to incorporate said mutations into a plasmid encoding the reference biomolecule. For biomolecule variants with a plurality of mutations, in some embodiments more than one oligonucleotide is used. In some embodiments, the DNA oligonucleotides encoding one or more mutations wherein the mutation region is flanked by between 10 and 100 nucleotides of homology to the target plasmid, both 5′ and 3′ to the mutation. Such oligonucleotides can in some embodiments be commercially synthesized and used in PCR amplification. An exemplary template for an oligonucleotide encoding a mutation is provided below:

-   -   5′-(N)₁₀₋₁₀₀-Mutation-(N′)₁₀₋₁₀₀-3′

In this exemplary oligonucleotide design, the Ns represent a sequence identical to the target plasmid, referred to herein as the homology arms. When a particular monomer in the biomolecule is targeted for mutation, these homology arms directly flank the DNA encoding the monomer in the target plasmid. In some exemplary embodiments where the biomolecule undergoing DME is a protein, 40 different oligonucleotides, using the same set of homology arms, are used to encode the enumerated 40 different amino acid mutations for each amino acid residue in the protein that is targeted for DME. When the mutation is of a single amino acid, the region encoding the desired mutation or mutations comprises three nucleotides encoding an amino acid (for substitutions or single insertions), or zero nucleotides (for deletions). In some embodiments, the oligonucleotide encodes insertion of greater than one amino acid. For example, wherein the oligonucleotide encodes the insertion of X amino acids, the region encoding the desired mutation comprises 3*X nucleotides encoding the X amino acids. In some embodiments, the mutation region encodes more than one mutation, for example mutations to two or more monomers of a biomolecule that are in close proximity (e.g., next to each other, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more monomers of each other).

Nucleotide sequences code for particular amino acid monomers in a substitution or insertion mutation in an oligo as described herein will be known to the person of ordinary skill in the art. For example, TTT or TTC triplets can be used to encode phenylalanine; TTA, TTG, CTT, CTC, CTA or CTG can be used to encode leucine; ATT, ATC or ATA can be used to encode isoleucine; ATG can be used to encode methionine; GTT, GTC, GTA or GTG c can be used to encode valine; TCT, TCC, TCA, TCG, AGT or AGC can be used to encode serine; CCT, CCC, CCA or CCG can be used to encode proline; ACT, ACC, ACA or ACG can be used to encode threonine; GCT, GCC, GCA or GCG can be used to encode alanine; TAT or TAC can be used to encode tyrosine; CAT or CAC can be used to encode histidine; CAA or CAG can be used to encode glutamine, AAT or AAC can be used to encode asparagine; AAA or AAG can be used to encode lysine; GAT or GAC can be used to encode aspartic acid; GAA or GAG can be used to encode glutamic acid; TGT or TGC c can be used to encode cysteine; TGG can be used to encode tryptophan; CGT, CGC, CGA, CGG, AGA or AGG can be used to encode arginine; and GGT, GGC, GGA or GGG can be used to encode glycine. In addition, ATG is used for initiation of the peptide synthesis as well as for methionine and TAA, TAG and TGA can be used to encode for the termination of the peptide synthesis.

In some exemplary embodiments where the biomolecule undergoing DME is an RNA, 8 different oligonucleotides, using the same set of homology arms, encode the above enumerated 8 different single nucleotide mutations for each nucleotide in the RNA that is targeted for DME. When the mutation is of a single ribonucleotide, the region of the oligo encoding the mutations can consist of the following nucleotide sequences: one nucleotide specifying a nucleotide (for substitutions or insertions), or zero nucleotides (for deletions). In some embodiments, the oligonucleotides are synthesized as single stranded DNA oligonucleotides. In some embodiments, all oligonucleotides targeting a particular amino acid or nucleotide of a biomolecule subjected to DME are pooled. In some embodiments, all oligonucleotides targeting a biomolecule subjected to DME are pooled. There is no limit to the type or number of mutations that can be created simultaneously in a DME library.

c. DME Library Construction

In some embodiments, plasmid recombineering is utilized to construct one or more DME libraries. Plasmid recombineering is described in Higgins, Sean A., Sorel V. Y. Ouonkap, and David F. Savage (2017) “Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering” ACS Synthetic Biology, the contents of which are incorporated herein by reference in their entirety.

An exemplary library construction protocol shown below:

Day 1: A bla, bio-, lambda-Red1, mutS-, cmR E. coli strain (for example, EcNR2, Addgene ID: 26931) is streaked out on a LB agar plate containing standard concentrations of the antibiotics Chloramphenicol and Ampicillin. Colonies are grown overnight at 30° C.

Day 2: A single colony of EcNR2 is picked into 5 mL of LB liquid media containing standard concentrations of the antibiotics Chloramphenicol and Ampicillin. The culture is grown overnight with shaking at 30° C.

Day 3: Electrocompetent cells are made using any method known in the art. An non-limiting, exemplary protocol for making electrocompetent cells comprises:

(1) Dilute 50 uL of the overnight culture into 50 mL of LB liquid media containing standard concentrations of the antibiotics Chloramphenicol and Ampicillin. Grow this 50 mL culture with shaking at 30° C.

(2) Once the 50 mL culture has grown to an OD600=0.5, transfer to shaking growth at 42° C. in a liquid water bath. Care should be taken to limit this growth at 42° C. to 15 minutes.

(3) After heated growth, transfer the culture to an ice water bath and swirl for at least one minute to cool the culture.

(4) Pellet the culture by spinning at 4,000×g for 10 minutes. Decant the supernatant.

(5) Carefully wash and re-suspend the pellet by adding ice cold water up to 50 mL. Repeat spin step 4.

(6) Resuspend the pellet in 1 mL of ice cold water. The cells are now competent for a standard electroporation step.

The electrocompetent E. coli are then transformed with the DME oligonucleotides:

(1) Pooled DME oligonucleotides are diluted in water to a final concentration of 20 μM. If more than one mutation is to be generated simultaneously, the corresponding oligonucleotides should be combined and mixed thoroughly.

(2) Pure target plasmid, for example, from a miniprep, is diluted in water to a final concentration of 10 ng per μL.

(3) Mix on ice:

-   -   2.5 μL DME oligonucleotide mixture     -   1 μL target plasmid     -   46.5 μL electrocompetent EcNR2 cells

(4) Transfer the mixture to a sterile 0.1 cm electroporation cuvette on ice and perform an electroporation. For example, the parameters of 1800 kV, 200 Ω, 25 μF can be used.

(5) Recover the electroporated cells by adding 1 mL of standard warm SOC media. Grow the culture for one hour with shaking at 30° C.

(6) After the recovery, add 4 mL of additional standard LB media to the culture. Add Kanamycin antibiotic at standard concentrations in order to select for the electroporated target plasmid. The culture is then grown=overnight with shaking at 30° C.

Day 4. Methods of isolating the target plasmid from overnight cultures will be readily apparent to one of ordinary skill in the art. For example, target plasmid can be isolated using commercial MiniPrep kits such as the MiniPrep kit from Qiagen. The plasmid library obtained comprises mutated target plasmids. In some embodiments, the plasmid library comprises between 10% and 30% mutated target plasmids. Additional mutations can be progressively added by repeatedly passing the library through rounds of electroporation and outgrowth, with no practical limit on the number of rounds that may be performed. Thus, for example, in some embodiments the library comprises plasmids encoding greater than one mutation per plasmid. For example, in some embodiments the library comprises plasmids independently comprising one, two, three, four, five, six, seven eight, nine, or greater mutations per plasmid. In some embodiments, plasmids that do not comprise any mutations are also present (e.g., plasmids which did not incorporate a DME oligonucleotide).

In other embodiments, methods other than plasmid recombineering are used to construct one or more DME libraries, or a combination of plasmid recombineering and other methods are used to construct one or more DME libraries. For example, DME libraries may, in some embodiments, be constructed using one of the other mutational methods described herein. Such libraries may then be taken through the library screening as described herein, and further iterations be carried out if desired.

d. Library Screening

Any appropriate method for screening or selecting a DME library is envisaged as following within the scope of the inventions. High throughput methods may be used to evaluate large libraries with thousands of individual mutations. In some embodiments, the throughput of the library screening or selection assay has a throughput that is in the millions of individual cells. In some embodiments, assays utilizing living cells are preferred, because phenotype and genotype are physically linked in living cells by nature of being contained within the same lipid bilayer. Living cells can also be used to directly amplify sub-populations of the overall library. In other embodiments, smaller assays are used in DME methods, for example to screen a focused library developed through multiple rounds of mutation and evaluation. Exemplary methods of screening libraries are described in Examples 24 and 25.

An exemplary, but non-limiting DME screening assay comprises Fluorescence-Activated Cell Sorting (FACS). In some embodiments, FACS may be used to assay millions of unique cells in a DME library. An exemplary FACS screening protocol comprises the following steps:

(1) PCR amplifying the purified plasmid library from the library construction phase. Flanking PCR primers can be designed that add appropriate restriction enzyme sites flanking the DNA encoding the biomolecule. Standard oligonucleotides can be used as PCR primers, and can be synthesized commercially. Commercially available PCR reagents can be used for the PCR amplification, and protocols should be performed according to the manufacturer's instructions. Methods of designing PCR primers, choice of appropriate restriction enzyme sites, selection of PCR reagents and PCR amplification protocols will be readily apparent to the person of ordinary skill in the art.

(2) The resulting PCR product is digested with the designed flanking restriction enzymes. Restriction enzymes may be commercially available, and methods of restriction enzyme digestion will be readily apparent to the person of ordinary skill in the art.

(3) The PCR product is ligated into a new DNA vector. Appropriate DNA vectors may include vectors that allow for the expression of the DME library in a cell. Exemplary vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors and plasmids. This new DNA vector can be part of a protocol such as lentiviral integration in mammalian tissue culture, or a simple expression method such as plasmid transformation in bacteria. Any vectors that allow for the expression of the biomolecule, and the DME library of variants thereof, in any suitable cell type, are considered within the scope of the disclosure. Cell types may include bacterial cells, yeast cells, and mammalian cells. Exemplary bacterial cell types may include E. coli. Exemplary yeast cell types may include Saccharomyces cerevisiae. Exemplary mammalian cell types may include mouse, hamster, and human cell lines, such as HEK293 cells, HEK293T cells, HEK293-F cells, Lenti-X 293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, A549 cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS, WI38 cells, MRC5 cells, HeLa, HT1080 cells, or CHO cells. Choice of vector and cell type will be readily apparent to the person of ordinary skill in the art. DNA ligase enzymes can be purchased commercially, and protocols for their use will also be readily apparent to one of ordinary skill in the art.

(4) Once the DME library has been cloned into a vector suitable for in vivo expression, the DME library is screened. If the biomolecule has a function which alters fluorescent protein production in a living cell, the biomolecule's biochemical function will be correlated with the fluorescence intensity of the cell overall. By observing a population of millions of cells on a flow cytometer, a DME library can be seen to produce a broad distribution of fluorescence intensities. Individual sub-populations from this overall broad distribution can be extracted by FACS. For example, if the function of the biomolecule is to repress expression of a fluorescent protein, the least bright cells will be those expressing biomolecules whose function has been improved by DME. Alternatively, if the function of the biomolecule is to increase expression of a fluorescent protein, the brightest cells will be those expressing biomolecules whose function has been improved by DME. Cells can be isolated based on fluorescence intensity by FACS and grown separately from the overall population. An exemplary FACS screening assay is shown in FIG. 2.

(5) After FACS sorting cells expressing a DME library of biomolecule variants, cultures comprising the original DME library and/or only highly functional biomolecule variants, as determined by FACS sorting, can be amplified separately. If the cells that were FACS sorted comprise cells that express the DME library of biomolecule variants from a plasmid (for example, E. coli cells transformed with a plasmid expression vector), these plasmids can be isolated, for example through miniprep. Conversely if the DME library of biomolecule variants has been integrated into the genomes of the FACs sorted cells, this DNA region can be PCR amplified and, optionally, subcloned into a suitable vector for further characterization using methods known in the art. Thus, the end product of library screening is a DNA library representing the initial, or ‘naive’, DME library, as well as one or more DNA libraries containing sub-populations of the naive DME library, which comprise highly functional mutant variants of the biomolecule identified by the screening processes described herein.

In some embodiments, DME libraries that have been screened or selected for highly functional variants are further characterized. In some embodiments, further characterizing the DME library comprises analyzing DME variants individually through sequencing, such as Sanger sequencing, to identify the specific mutation or mutations that gave rise to the highly functional variant. Individual mutant variants of the biomolecule can be isolated through standard molecular biology techniques for later analysis of function. In some embodiments, further characterizing the DME library comprises high throughput sequencing of both the naive library and the one or more libraries of highly functional variants. This approach may, in some embodiments, allow for the rapid identification of mutations that are over-represented in the one or more libraries of highly functional variants compared to the naive DME library. Without wishing to be bound by any theory, mutations that are over-represented in the one or more libraries of highly functional variants are likely to be responsible for the activity of the highly functional variants. In some embodiments, further characterizing the DME library comprises both sequencing of individual variants and high throughput sequencing of both the naive library and the one or more libraries of highly functional variants.

High throughput sequencing can produce high throughput data indicating the functional effect of the library members. In embodiments wherein one or more libraries represents every possible mutation of every monomer location, such high throughput sequencing can evaluate the functional effect of every possible DME mutation. Such sequencing can also be used to evaluate one or more highly functional sub-populations of a given library, which in some embodiments may lead to identification of mutations that result in improved function. An exemplary protocol for high throughput sequencing of a library with a highly functional sub-population is as follows:

(1) High throughput sequencing of the Naive DME library, N. High throughput sequence the highly functional sub-population library. F. Any high throughput sequencing platform that can generate a suitable abundance of reads can be used. Exemplary sequencing platforms include, but are not limited to Illumina, Ion Torrent, 454 and PacBio sequencing platforms.

(2) Select a particular mutation to evaluate, i. Calculate the total fractional abundance of i in N, i(N). Calculate the total fractional abundance of i in F, i(F).

(3) Calculate the following: [(i(F)+1)/(i(N)+1)]. This value, the ‘enrichment ratio’, is correlated with the function of the particular mutant variant i of the biomolecule.

(4) Calculate the enrichment ratio for each of the mutations observed in deep sequencing of the DME libraries.

(5) The set of enrichment ratios for the entire library can be converted to a log scale such that a value of zero represents no enrichment (i.e. an enrichment ratio of one), values greater than zero represent enrichment, and values less than zero represent depletion. Alternatively, the log scale can be set such that 1.5 represents enrichment, and −0.6 represents depletion, as in FIG. 3A, FIG. 3B, FIG. 4A, FIG. 4C. These rescaled values can be referred to as the relative ‘fitness’ of any particular mutation. These fitness values quantitatively indicate the effect a particular mutation has on the biochemical function of the biomolecule.

(6) The set of calculated DME fitness values can be mapped to visually represent the fitness landscape of all possible mutations to a biomolecule. The fitness values can also be rank ordered to determine the most beneficial mutations contained within the DME library.

e. Iterating DME

In some embodiments, a highly functional variant produced by DME has more than one mutation. For example, combinations of different mutations can in some embodiments produce optimized biomolecules whose function is further improved by the combination of mutations. In some embodiments, the effect of combining mutations on function of the biomolecule is linear. As used herein, a combination of mutations that is linear refers to a combination whose effect on function is equal to the sum of the effects of each individual mutation when assayed in isolation. In some embodiments, the effect of combining mutations on function of the biomolecule is synergistic. As used herein, a combination of mutations that is synergistic refers to a combination whose effect on function is greater than the sum of the effects of each individual mutation when assayed in isolation. Other mutations may exhibit additional unexpected nonlinear additive effects, or even negative effects. This phenomenon is known as epistasis.

Epistasis can be unpredictable, and is a significant source of variation when combining mutations. Epistatic effects can be addressed through additional high throughput experimental methods in DME library construction and assay. In some embodiments, the entire DME protocol can be iterated, returning to the library construction step and selecting only mutations identified as having desired effects (such as increased functionality) from an initial DME library screen. Thus, in some embodiments, DME library construction and screening is iterated, with one or more cycles focusing the library on a subset of mutations having desired effects. In such embodiments, layering of selected mutations may lead to improved variants. In some alternative embodiments, DME can be repeated with the full set of mutations, but targeting a novel, pre-mutated version of the biomolecule. For example, one or more highly functional variants identified in a first round of DME library construction, assay, and characterization can be used as the target plasmid for further rounds of DME using a broad, unfocused set of further mutations (such as every possible mutation, or a subset thereof), and the process repeated. Any number, type of iterations or combinations of iterations of DME are envisaged as within the scope of the disclosure.

f. Deep Mutational Scanning

In some embodiments, Deep Mutational Scanning (DMS) is used to identify CasX variant proteins with improved function. Deep mutational scanning assesses protein plasticity as it relates to function. In DMS methods, every amino acid of a protein is changed to every other amino acid and absolute protein function assayed. For example, every amino acid in a CasX protein can be changed to every other amino acid, and the mutated CasX proteins assayed for their ability to bind to or cleave DNA. Exemplary assays such as the CRISPRi assay or bacterial-based cleavage assays that can be used to characterize collections of DMS CasX variant proteins are described in Oakes et al. (2016) “Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch” Nat Biotechnol 34(6):646-51 and Liu et al. (2019) “CasX enzymes comprise a distinct family of RNA-guided genome editors” Nature doi.org/10.1038/s41586-019-0908; the contents of which are incorporated herein by reference.

In some embodiments, DMS is used to identify CasX proteins with improved DNA binding activity. In some embodiments, DNA binding activity is assayed using a CRISPRi assay. In a non-limiting, exemplary embodiment of a CRISPRi assay, cells expressing a fluorescent protein such as green fluorescent protein (GFP) or red fluorescent protein (RFP) are assayed using FACS to identify CasX variants capable of repressing expression of the fluorescent protein in a sgNA dependent fashion. In this example, a catalytically dead CasX (dCasX) is used to generate the collection of DMS mutants being assayed. The wild-type CasX protein binds to its cognate sgNA and forms a protein-RNA complex. The complex binds to specific DNA targets by Watson-Crick base pairing between the sgNA and the DNA target, in this case a DNA sequence encoding the fluorescent protein. In the case of wild-type CasX, the DNA will be cleaved due to the nuclease activity of the CasX protein. However, without wishing to be bound by theory, it is likely that dCasX is still able to form a complex with the sgNA and bind to specific DNA target. When targeting of dCasX occurs to the protein-coding region, it blocks RNA polymerase II and transcript initiation and/or elongation, leading to a reduction in fluorescent protein expression that can be detected by FACs.

In some embodiments, DMS is used to identify CasX proteins with improved DNA cleavage activity. Methods of assaying the DNA cleavage efficiency of CasX variant proteins will be apparent to one of ordinary skill in the art. For example, CasX proteins complexed with an sgNA with a spacer complementary to a particular target DNA sequence can be used to cleave the DNA target sequence in vitro or in vivo in a suitable cell type, and the frequency of insertions and deletions at the site of cleavage are assayed. Without wishing to be bound by theory, cleavage or nicking by CasX generates double-strand breaks in DNA, whose subsequent repair by the non-homologous end joining pathway (NHEJ) gives rise to small insertions or deletions (indels) at the site of the double-strand breaks. The frequency of indels at the site of CasX cleavage can be measured using high throughput or Sanger sequencing of the target sequence. Alternatively, or in addition, frequency of indel generation by CasX cleavage of a target sequence can be measured using mismatch assays such as T7 Endonuclease I (17EI) or Surveyor mismatch assays.

In some embodiments, following DMS, a map of the genotypes of DMS mutants linked with their resulting phenotype (for example, a heat map) is generated and used to characterize fundamental principles of the protein. All possible mutations are characterized as leading to functional or nonfunctional protein products to establish that protein's functional landscape.

g. Error Prone PCR

In some embodiments, Error Prone PCR is used to generate CasX protein or sgNA scaffold variants with improved function. Polymerases that replicate DNA have different levels of fidelity. One way of introducing random mutations to a gene is through an error prone polymerase that will incorporate incorrect nucleotides at a range of frequencies. This frequency can be modulated depending on the desired outcome. In some embodiments, a polymerase and conditions for polymerase activity are selected that result in a frequency of nucleotide changes that produces an average of n 1-4 amino acid changes in a protein sequence. An exemplary error prone polymerase comprises Agilent's GeneMorphII kit. The GeneMorphII kit can be used to amplify a DNA sequence encoding a wild type CasX protein (for example, a protein of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), according to the manufacturer's protocol, thereby subjecting the protein to unbiased random mutagenesis and generating a diverse population of CasX variant proteins. This diverse population of CasX variant proteins can then be assayed using the same assays described above for DMS to observe how changes in genotype relate to changes in phenotype.

h. Cassette Mutagenesis

In some embodiments, cassette mutagenesis is used to generate CasX variant protein or sgNA scaffold variants with improved function. Cassette mutagenesis takes advantage of unique restriction enzyme sites that are replaced by degenerative nucleotides to create small regions of high diversity in select areas of a gene of interest such as a CasX protein or sgNA scaffold. In an exemplary cassette mutagenesis protocol, restriction enzymes are used to cleave near the sequence targeted for mutagenesis on DNA molecule encoding a CasX protein or sgNA scaffold contained in a suitable vector. This step removes the sequence targeted for mutagenesis and everything between the restriction sites. Then, synthetic double stranded DNA molecules containing the desired mutation and ends that are complimentary to the restriction digest ends are ligated in place of the sequence that has been removed by restriction digest, and suitable cells, such as E. coli are transformed with the ligated vector. In some embodiments, cassette mutagenesis can be used to generate one or more specific mutations in a CasX protein or sgNA scaffold. In some embodiments, cassette mutagenesis can be used to generate a library of CasX variant proteins or sgNA scaffold variants that can be screened or selected for improved function using the methods described herein. For example, in using cassette mutagenesis to generate CasX variants, parts of the Non-Target Strand Binding (NTSB) domain can be replaced with a sequence of degenerate nucleotides. Sequences of degenerate nucleotides can be highly localized to regions of the CasX protein, for example regions of the NTSB that are of interest because of their highly mobile elements or their direct contacts with DNA. Libraries of CasX variant proteins generated via cassette mutagenesis can then be screened using the assays described herein for DME. DMS and error prone PCR and variants can be selected for improved function.

i. Random Mutagenesis

In some embodiments, random mutagenesis is used to generate CasX variant proteins or sgNA scaffold variants with improved function. Random mutagenesis is an unbiased way of changing DNA. Exemplary methods of random mutagenesis will be known to the person of ordinary skill in the art and include exposure to chemicals. UV light, X-rays or use of unstable cell lines. Different mutagenic agents produce different types of mutations, and the ordinarily skilled artisan will be able to select the appropriate agent to generate the desired type of mutations. For example, ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can be used to generate single base pair changes, while X-rays often result in deletions and gross chromosomal rearrangements. UV light exposure produces dimers between adjacent pyrimidines in DNA, which can result in point mutations, deletions and rearrangements. Error prone cell lines can also be used to introduce mutations, for example on a plasmid comprising a CasX protein or sgNA scaffold of the disclosure. A population of DNA molecules encoding a CasX protein (for example, a protein of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) or an sgNA scaffold can be exposed to a mutagen to generate collection of CasX variant proteins or sgNA scaffold variants, and these collections can be assayed for improved function using any of the assays described herein.

j. Staggered Extension Process (StEP)

In some embodiments, a staggered extension process (StEP) is used to generate CasX variant proteins or sgNA scaffold variants with improved function. Staggered extension process is a specialized PCR protocol that allows for the breeding of multiple variants of a protein during a PCR reaction. StEP utilizes a polymerase with low processivity, (for example Taq or Vent polymerase) to create short primers off of two or more different template strands with a significant level of sequence similarity. The short primers are then extended for short time intervals allowing for shuffling of the template strands. This method can also be used as a means to stack DME variants. Exemplary StEP protocols are described by Zhao, H. et al. (1998) “Molecular evolution by staggered extension process (StEP) in vitro recombination” Nature Biotechnology 16: 258-261, the contents of which are incorporated herein by reference in their entirety. StEP can be used to generate collections of CasX variant proteins or sgNA scaffold variants, and these collections can be assayed for improved function using any of the assays described herein.

k. Gene Shuffling

In some embodiments, gene shuffling is used to generate CasX variant proteins or sgNA scaffold variants with improved function. In some embodiments, gene shuffling is used to combine (sometimes referred to herein as “stack”) variants produced through other methods described herein, such as plasmid recombineering. In an exemplary gene shuffling protocol, a DNase, for example DNase I, is used to shear a set of parent genes into pieces of 50-100 base pair (bp) in length. In some embodiments, these parent genes comprise CasX variant proteins with improved function created and isolated using the methods described herein. In some embodiments, these parent genes comprise sgNA scaffold variants with improved function created and isolated using the methods described herein. Dnase fragmentation is then followed by a polymerase chain reaction (PCR) without primers. DNA fragments with sufficient overlapping homologous sequence will anneal to each other and are then extended by DNA polymerase. If different fragments comprising different mutations anneal, the result is a new variant combining those two mutations. In some embodiments, PCR without primers is followed by PCR extension, and purification of shuffled DNA molecules that have reached the size of the parental genes (e.g., a sequence encoding a CasX protein or sgNA scaffold). These genes can then be amplified with another PCR, for example by adding PCR primers complementary to the 5′ and 3′ ends of gene undergoing shuffling. In some embodiments, the primers may have additional sequences added to their 5′ ends, such as sequences for restriction enzyme recognition sites needed for ligation into a cloning vector.

l. Domain Swapping

In some embodiments, domain swapping is used to generate CasX variant proteins or sgNA scaffold variants with improved function. To generate CasX variant proteins, engineered domain swapping can be used to mix and match parts with other proteins and CRISPR molecules. For example, CRISPR proteins have conserved RuvC domains, so the CasX RuvC domain could be swapped for that of other CRISPR proteins, and the resulting protein assayed for improved DNA cleavage using the assays described herein. For sgNAs, the scaffold stem, extended stem or loops can be exchanged with structures found in other RNAs, for example the scaffold stem and extended stem of the sgNA can be exchanged with thermostable stem loops from other RNAs, and the resulting variant assayed for improved function using the assays described herein. In some embodiments, domain swapping can be used to insert new domains into the CasX protein or sgNA. In some exemplary embodiments where domain swapping is applied to a protein, the inserted domain comprises an entire second protein.

VII. Vectors

In some embodiments, provided herein are vectors comprising polynucleotides encoding the CasX variant proteins and sgNA or dgNA variants and, optionally, donor template polynucleotides, described herein. In some cases, the vectors are utilized for the expression and recovery of the CasX, gNA (and, optionally, the donor template) components of the gene editing pair. In other cases, the vectors are utilized for the delivery of the encoding polynucleotides to target cells for the editing of the target nucleic acid, as described more fully, below.

In some embodiments, provided herein are polynucleotides encoding the sgNA or dgNA variants described herein. In some embodiments, said polynucleotides are DNA. In other embodiments, said polynucleotides are RNA. In some embodiments, provided herein are vectors comprising the polynucleotides sequences encoding the sgNA or dgNA variants described herein. In some embodiments, the vectors comprising the polynucleotides include bacterial plasmids, viral vectors, and the like. In some embodiments, a CasX variant protein and a sgNA variant are encoded on the same vector. In some embodiments, a CasX variant protein and a sgNA variant are encoded on different vectors.

In some embodiments, the disclosure provides a vector comprising a nucleotide sequence encoding the components of the CasX:gNA system. For example, in some embodiments provided herein is a recombinant expression vector comprising a) a nucleotide sequence encoding a CasX variant protein; and b) a nucleotide sequence encoding a gNA variant described herein. In some cases, the nucleotide sequence encoding the CasX variant protein and/or the nucleotide sequence encoding the gNA variant are operably linked to a promoter that is operable in a cell type of choice (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a primate cell, a rodent cell, a human cell). Suitable promoters for inclusion in the vectors are described herein, below.

In some embodiments, the nucleotide sequence encoding the CasX variant protein is codon optimized. This type of optimization can entail a mutation of a CasX-encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized CasX variant-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized CasX variant-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a plant cell, then a plant codon-optimized CasX variant protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a bacterial cell, then a bacterial codon-optimized CasX variant protein-encoding nucleotide sequence could be generated.

In some embodiments, provided herein are one or more recombinant expression vectors such as (i) a nucleotide sequence of a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome); (ii) a nucleotide sequence that encodes a gNA or a gNA variant as described herein, that may be provided in a single-guide or dual-guide form, (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (iii) a nucleotide sequence encoding a CasX protein or a CasX variant protein (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). In some embodiments, the sequences encoding the gNA and CasX proteins are in different recombinant expression vectors, and in other embodiments the gNA and CasX proteins are in the same recombinant expression vector. In some embodiments, the sequences encoding the gNA, the CasX protein, and the donor template(s) are in different recombinant expression vectors, and in other embodiments one or more are in the same recombinant expression vector. In some embodiments, either the sgNA in the recombinant expression vector, the CasX protein encoded by the recombinant expression vector, or both, are variants of a reference CasX protein or gNAs as described herein. In the case of the nucleotide sequence encoding the gNA, the recombinant expression vector can be transcribed in vitro, for example using T7 promoter regulatory sequences and T7 polymerase in order to produce the gRNA, which can then be recovered by conventional methods; e.g., purification via gel electrophoresis. Once synthesized, the gRNA may be utilized in the gene editing pair to directly contact a target DNA or may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

In some embodiments, a nucleotide sequence encoding a reference or variant CasX and/or gNA is operably linked to a control element; e.g., a transcriptional control element, such as a promoter. In some embodiments, a nucleotide sequence encoding a reference or CasX variant protein is operably linked to a control element; e.g., a transcriptional control element, such as a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, etc.). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 10 fold, by 100 fold, more usually by 1000 fold.

Non-limiting examples of eukaryotic promoters (promoters functional in a eukaryotic cell) include EF1alpha, EF1alpha core promoter, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Further non-limiting examples of eukaryotic promoters include the CMV promoter full-length promoter, the minimal CMV promoter, the chicken β-actin promoter, the hPGK promoter, the HSV TK promoter, the Mini-TK promoter, the human synapsin I promoter which confers neuron-specific expression, the Mecp2 promoter for selective expression in neurons, the minimal IL-2 promoter, the Rous sarcoma virus enhancer/promoter (single), the spleen focus-forming virus long terminal repeat (LTR) promoter, the SV40 promoter, the SV40 enhancer and early promoter, the TBG promoter: promoter from the human thyroxine-binding globulin gene (Liver specific), the PGK promoter, the human ubiquitin C promoter, the UCOE promoter (Promoter of HNRPA2B1-CBX3), the Histone H2 promoter, the Histone H3 promoter, the U1a1 small nuclear RNA promoter (226 nt), the U1b2 small nuclear RNA promoter (246 nt) 26, the TTR minimal enhancer/promoter, the b-kinesin promoter, the human eIF4A1 promoter, the ROSA26 promoter and the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter.

Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6×His tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to the CasX protein, thus resulting in a chimeric CasX polypeptide.

In some embodiments, a nucleotide sequence encoding a gNA variant and/or a CasX variant protein is operably linked to a promoter that is an inducible promoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein) or a promoter that is a constitutively active promoter (i.e., a promoter that is constitutively in an active/“ON” state). In other embodiments, a nucleotide sequence encoding a gNA variant and/or a CasX variant protein is operably linked to a spatially restricted promoter (i.e., transcriptional control element, enhancer, tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

In certain embodiments, suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, a human HI promoter (HI), a POL1 promoter, a 7SK promoter, tRNA promoters and the like.

In some embodiments, a nucleotide sequence encoding a gNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an HI promoter, and the like). As would be understood by one of ordinary skill in the art, when expressing an RNA (e.g., a gRNA) from a nucleic acid (e.g., an expression vector) using a U6 promoter (e.g., in a eukaryotic cell), or another PolIII promoter, the RNA may need to be mutated if there are several Ts in a row (coding for Us in the RNA). This is because a string of Ts (e.g., 5 Ts) in DNA can act as a terminator for polymerase III (Pol III). Thus, in order to ensure transcription of a gRNA (e.g., the activator portion and/or targeter portion, in dual guide or single guide format) in a eukaryotic cell, it may sometimes be necessary to modify the sequence encoding the gRNA to eliminate runs of Ts. In some cases, a nucleotide sequence encoding a CasX protein (e.g., a wild type CasX protein, a nickase CasX protein, a dCasX protein, a chimeric CasX protein and the like) is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EF1alpha promoter, an estrogen receptor-regulated promoter, and the like).

In certain embodiments, inducible promoters suitable for use may include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracyclinc (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

In some cases, the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell; prokaryotic cell).

In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including Tet Activators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

Recombinant expression vectors of the disclosure can also comprise elements that facilitate robust expression of reference or CasX variant proteins and/or reference or variant gNAs of the disclosure. For example, recombinant expression vectors can include one or more of a polyadenylation signal (PolyA), an intronic sequence or a post-transcriptional regulatory element such as a woodchuck hepatitis post-transcriptional regulatory element (WPRE). Exemplary polyA sequences include hGH poly(A) signal (short), HSV TK poly(A) signal, synthetic polyadenylation signals, SV40 poly(A) signal, s3-globin poly(A) signal and the like. In addition, vectors used for providing a nucleic acid encoding a gNA and/or a CasX protein to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the gNA and/or CasX protein. A person of ordinary skill in the art will be able to select suitable elements to include in the recombinant expression vectors described herein.

A recombinant expression vector sequence can be packaged into a virus or virus-like particle (also referred to herein as a “particle” or “virion”) for subsequent infection and transformation of a cell, ex vivo, in vitro or in vivo. Such particles or virions will typically include proteins that encapsidate or package the vector genome. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

Adeno-associated virus (AAV) is a small (20 nm), nonpathogenic virus that is useful in treating human diseases in situations that employ a viral vector for delivery to a cell such as a eukaryotic cell, either in vivo or ex vivo for cells to be prepared for administering to a subject. A construct is generated, for example a construct encoding any of the CasX proteins and/or gNA embodiments as described herein, and is flanked with AAV inverted terminal repeat (ITR) sequences, thereby enabling packaging of the AAV vector into an AAV viral particle.

An “AAV” vector may refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise. As used herein, the term “serotype” refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., there are many known serotypes of primate AAVs. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV-Rh74 (Rhesus macaque-derived AAV), and AAVRh10, and modified capsids of these serotypes. For example, serotype AAV-2 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV-2 and a genome containing 5′ and 3′ ITR sequences from the same AAV-2 serotype. Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5′-3′ ITRs of a second serotype. Pseudotyped rAAV would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. Pseudotyped recombinant AAV (rAAV) are produced using standard techniques described in the art. As used herein, for example, rAAV1 may be used to refer an AAV having both capsid proteins and 5′-3′ ITRs from the same serotype or it may refer to an AAV having capsid proteins from serotype 1 and 5′-3′ ITRs from a different AAV serotype, e.g., AAV serotype 2. For each example illustrated herein the description of the vector design and production describes the serotype of the capsid and 5′-3′ ITR sequences.

An “AAV virus” or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle additionally comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome to be delivered to a mammalian cell), it is typically referred to as “rAAV”. An exemplary heterologous polynucleotide is a polynucleotide comprising a CasX protein and/or sgRNA and, optionally, a donor template of any of the embodiments described herein.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” is meant the art recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, for example Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridae and their Replication” in Fundamental Virology, 2^(nd) Edition, (B. N. Fields and D. M. Knipe, eds.). As used herein, an AAV ITR need not have the wild-type nucleotide sequence depicted, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, and AAVRh10, and modified capsids of these serotypes. Furthermore, 5′ and 3′ ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Use of AAV serotypes for integration of heterologous sequences into a host cell is known in the art (see, e.g., WO2018195555A1 and US20180258424A1, incorporated by reference herein).

By “AAV rep coding region” is meant the region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome. By “AAV cap coding region” is meant the region of the AAV genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These Cap expression products supply the packaging functions which are collectively required for packaging the viral genome.

In some embodiments, AAV capsids utilized for delivery of the encoding sequences for the CasX and gNA, and, optionally, the donor template nucleotides to a host cell can be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74 (Rhesus macaque-derived AAV), and AAVRh10, and the AAV ITRs are derived from AAV serotype 2.

In order to produce rAAV viral particles, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. Packaging cells are typically used to form virus particles; such cells include HEK293 cells (and other cells known in the art), which package adenovirus. A number of transfection techniques are generally known in the art; see, e.g., Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transfection methods include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles.

In some embodiments, host cells transfected with the above-described AAV expression vectors are rendered capable of providing AAV helper functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV viral particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors. Thus, AAV helper functions include one, or both of the major AAV ORFs (open reading frames), encoding the rep and cap coding regions, or functional homologues thereof. Accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art. Commonly, accessory functions are provided by infection of the host cells with an unrelated helper virus. In some embodiments, accessory functions are provided using an accessory function vector. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., may be used in the expression vector.

In other embodiments, retroviruses, for example, lentiviruses, may be suitable for use as vectors for delivery of the encoding nucleic acids of the CasX:gNA systems of the present disclosure. Commonly used retroviral vectors are “defective”, e.g. unable to produce viral proteins required for productive infection, and may be referred to a virus-like particles (VLP). Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into VLP capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art.

For non-viral delivery, vectors can also be delivered wherein the vector or vectors encoding the CasX variants and gNA are formulated in nanoparticles, wherein the nanoparticles contemplated include, but are not limited to nanospheres, liposomes, quantum dots, polyethylene glycol particles, hydrogels, and micelles. Lipid nanoparticles are generally composed of an ionizable cationic lipid and three or more additional components, such as cholesterol, DOPE, polylactic acid-co-glycolic acid, and a polyethylene glycol (PEG) containing lipid. In some embodiments, the CasX variants of the embodiments disclosed herein are formulated in a nanoparticle. In some embodiments, the nanoparticle comprises the gNA of the embodiments disclosed herein. In some embodiments, the nanoparticle comprises RNP of the CasX variant complexed with the gNA. In some embodiments, the system comprises a nanoparticle comprising nucleic acids encoding the CasX variants and the gNA and, optionally, a donor template nucleic acid. In some embodiments, the components of the CasX:gNA system are formulated in separate nanoparticles for delivery to cells or for administration to a subject in need thereof.

VIII. Applications

The CasX proteins, guides, nucleic acids, and variants thereof provided herein, as well as vectors encoding such components, are useful for various applications, including therapeutics, diagnostics, and research.

Provided herein are methods of cleaving a target DNA, comprising contacting the target DNA with a CasX protein and gNA pair. In some embodiments, the pair comprises a CasX variant protein and a gNA, wherein the CasX variant protein is a CasX variant of SEQ ID NO: 2 as described herein (e.g., a sequence of Tables 3, 8, 9, 10 and 12), and wherein the contacting results in cleavage and, optionally, editing of the target DNA. In other embodiments, the pair comprises a reference CasX protein and a gNA. In some embodiments, the gNA is a gNA variant of the disclosure (e.g., a sequence of SEQ ID NOS: 2101-2280), or a reference gRNA scaffold comprising SEQ ID NO: 5 or SEQ ID NO: 4, and further comprises a spacer that is complementary to the target DNA.

In yet further aspects, the disclosure provides methods of cleaving a target DNA, comprising contacting the target DNA with a CasX protein and gNA pair of any of the embodiments described herein, wherein the contacting results in cleavage and optionally editing of the target DNA. In some embodiments, the scaffold of the gNA variant comprises a sequence of SEQ ID NO: 2101-2280, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto, and further comprises a spacer that is complementary to the target DNA. In some embodiments, the CasX protein is a CasX variant protein of any of the embodiments described herein (e.g., a sequence of Tables 3, 8, 9, 10 and 12), or a reference CasX protein SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, the methods of editing a target DNA comprise contacting a target DNA with a CasX protein and gNA pair as described herein and a donor polynucleotide, sometimes referred to as a donor template. In some embodiments, CasX protein and gNA pairs generate site-specific double strand breaks (DSBs) or single strand breaks (SSBs) (e.g., when the CasX variant protein is a nickase) within double-stranded DNA (dsDNA) target nucleic acids, which are repaired either by non-homologous end joining (NHEJ), homology-directed repair (HDR), homology-independent targeted integration, micro-homology mediated end joining (MMEJ), single strand annealing (SSA) or base excision repair (BER). In some cases, contacting a target DNA with a gene editing pair occurs under conditions that are permissive for NHEJ, HDR, or MMEJ. Thus, in some cases, a method as provided herein includes contacting the target DNA with a donor polynucleotide (e.g., by introducing the donor polynucleotide into a cell), wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. For example, an exogenous donor template which may comprise a corrective sequence (or a deletion to knock-out the defective allele) to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell. The upstream and downstream sequences relative to the cleavage site(s) share sequence similarity with either side of the site of integration in the target DNA (i.e., homologous arms), facilitating the insertion. In other cases, an exogenous donor template which may comprise a corrective sequence is inserted between the ends generated by CasX cleavage by homology-independent targeted integration (HITI) mechanisms. The exogenous sequence inserted by HITI can be any length, for example, a relatively short sequence of between 1 and 50 nucleotides in length, or a longer sequence of about 50-1000 nucleotides in length. The lack of homology can be, for example, having no more than 20-50% sequence identity and/or lacking in specific hybridization at low stringency. In other cases, the lack of homology can further include a criterion of having no more than 5, 6, 7, 8, or 9 bp identity. In some cases, the method does not comprise contacting a cell with a donor polynucleotide, and the target DNA is modified such that nucleotides within the target DNA are deleted or inserted according to the cells own repair pathways.

The donor template sequence may comprise certain sequence differences as compared to the genomic sequence, e.g., restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor nucleic acid at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). Alternatively, these sequence differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence. In some embodiments of the method, the donor polynucleotide comprises at least about 10, at least about 50, at least about 100, or at least about 200, or at least about 300, or at least about 400, or at least about 500, or at least about 600, or at least about 700, or at least about 800, or at least about 900, or at least about 1000, or at least about 10,000, or at least 15,000 nucleotides of a wild-type gene. In other embodiments, the donor polynucleotide comprises at least about 10 to about 15,000 nucleotides, or at least about 200 to about 10,000 nucleotides, or at least about 400 to about 6000 nucleotides, or at least about 600 to about 4000 nucleotides, or at least about 1000 to about 2000 nucleotides of a wild-type gene. In some embodiments, the donor template is a single stranded DNA template or a single stranded RNA template. In other embodiments, the donor template is a double stranded DNA template.

In some embodiments, contacting the target DNA with a CasX protein and gNA gene editing pair of the disclosure results in gene editing. In some embodiments, the editing occurs in vitro, outside of a cell, in a cell-free system. In some embodiments, the editing occurs in vitro, inside of a cell, for example in a cell culture system. In some embodiments, the editing occurs in vivo inside of a cell, for example in a cell in an organism. In some embodiments, the cell is a eukaryotic cell. Exemplary eukaryotic cells may include cells selected from the group consisting of a plant cell, a fungal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a pig cell, a dog cell, a primate cell, a non-human primate cell, and a human cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an embryonic stem cell, an induced pluripotent stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, an NK cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplated expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, fibroblasts, osteoblasts, chondrocytes, exogenous cell, endogenous cell, stem cell, hematopoietic stem cell, bone-marrow derived progenitor cell, myocardial cell, skeletal cell, fetal cell, undifferentiated cell, multi-potent progenitor cell, unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, or a post-natal stem cell. In alternative embodiments, the cell is a prokaryotic cell.

Methods of editing of the disclosure can occur in vitro outside of a cell, in vitro inside of a cell or in vivo inside of a cell. The cell can be in a subject. In some embodiments, editing occurs in the subject having a mutation in an allele of a gene wherein the mutation causes a disease or disorder in the subject. In some embodiments, editing changes the mutation to a wild type allele of the gene. In some embodiments, editing knocks down or knocks out expression of an allele of a gene causing a disease or disorder in the subject. In some embodiments, editing occurs in vitro inside of the cell prior to introducing the cell into a subject. In some embodiments, the cell is autologous or allogeneic.

Methods of introducing a nucleic acid (e.g., a nucleic acid comprising a donor polynucleotide sequence, one or more nucleic acids encoding a CasX protein and/or a gNA, or variants thereof as described herein) into a cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct such as an AAV or virus like particle (VLP; e.g. a capsid derived from one or more components of a retrovirus, described supra) vector comprising the encoded CasX and gNA components, as described, supra) into a cell. Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, nucleofection, electroporation, direct addition by cell penetrating CasX proteins that are fused to or recruit donor DNA, cell squeezing, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

Introducing recombinant expression vectors into cells can occur in any suitable culture media and under any suitable culture conditions that promote the survival of the cells. Introducing recombinant expression vectors into a target cell can be carried out in vivo, in vitro or ex vivo.

In some embodiments, a CasX variant protein can be provided as RNA. The RNA can be provided by direct chemical synthesis, or may be transcribed in vitro from a DNA (e.g., a DNA encoding an mRNA comprising a sequence encoding the CasX variant protein). Once synthesized, the RNA may, for example, be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection).

Nucleic acids may be provided to the cells using well-developed transfection techniques, and the commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit from Mirus Bio LLC, Lonza nucleofection, Maxagen electroporation and the like.

In some embodiments, vectors may be provided directly to a target host cell. For example, cells may be contacted with vectors comprising the subject nucleic acids (e.g., recombinant expression vectors having the donor template sequence and encoding the gNA variant, recombinant expression vectors encoding the CasX variant protein) such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids include electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising the subject viral expression vectors; e.g., the vectors are viral particles such as AAV or VLP that comprise polynucleotides that encode the CasX:gNA components or that comprise CasX:gNA RNP. For non-viral delivery, vectors or the CasX:gNA components can also be formulated for delivery in nanoparticles, wherein the nanoparticles contemplated include, but are not limited to nanospheres, liposomes, quantum dots, polyethylene glycol particles, hydrogels, and micelles.

A nucleic acid comprising a nucleotide sequence encoding a CasX variant protein is in some cases an RNA. Thus, in some embodiments a CasX variant protein can be introduced into cells as RNA. Methods of introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method used for the introduction of DNA. A CasX variant protein may instead be provided to cells as a polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like. Domains of interest may include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like. The polypeptide may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.

Additionally or alternatively, a reference or CasX variant protein of the present disclosure may be fused to a polypeptide permeant domain to promote uptake by the cell. A number of permeant domains are known in the art and may be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. For example, WO2017/106569 and US20180363009A1, incorporated by reference herein in its entirety, describe fusion of a Cas protein with one or more nuclear localization sequences (NLS) to facilitate cell uptake. In other embodiments, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 398). As another example, the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein. Other permeant domains include polyarginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like. The site at which the fusion is made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site will be determined by routine experimentation.

A CasX variant protein of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells transformed with encoding vectors (described above), and it may be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using methods known in the art. In the case of production of the gNA of the present disclosure, recombinant expression vectors encoding the gNA can be transcribed in vitro, for example using T7 promoter regulatory sequences and T7 polymerase in order to produce the gRNA, which can then be recovered by conventional methods; e.g., purification via gel electrophoresis. Once synthesized, the gRNA may be utilized in the gene editing pair to directly contact a target DNA or may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

In some embodiments, modifications of interest that do not alter the primary sequence of the CasX variant protein may include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

In other embodiments, the present disclosure provides nucleic acids encoding a gNA variant or encoding a CasX variant and reference CasX proteins that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

A CasX variant protein of the disclosure may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

A CasX variant protein of the disclosure may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise 50% or more by weight of the desired product, more usually 75% or more by weight, preferably 95% or more by weight, and for therapeutic purposes, usually 99.5% or more by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Thus, in some cases, a CasX polypeptide, or a CasX fusion polypeptide, of the present disclosure is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-CasX proteins or other macromolecules, etc.).

In some embodiments, to induce cleavage or any desired modification to a target nucleic acid (e.g., genomic DNA), or any desired modification to a polypeptide associated with target nucleic acid in an in vitro cell, the gNA variant and/or the CasX variant protein of the present disclosure and/or the donor template sequence, whether they be introduced as nucleic acids or polypeptides, are provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which may be repeated with a frequency of about every day to about every 7 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every 7 days. The agent(s) may be provided to the subject cells one or more times, e.g. one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event; e.g., 16-24 hours, after which time the media is replaced with fresh media and the cells are cultured further.

In some embodiments, the disclosure provides methods of treating a disease in a subject in need thereof comprising modifying a gene in a cell of the subject, the modifying comprising: a) administering to the subject a CasX protein of any of the embodiments described herein and a gNA of any of the embodiments described herein wherein the targeting sequence of the gNA has a sequence that hybridizes with the target nucleic acid; b) a nucleic acid encoding the CasX protein and gNA of any of the embodiments described herein; c) a vector comprising the nucleic acids encoding the CasX and gNA; d) a VLP comprising a CasX:gNA RNP; or e) combinations thereof. In some embodiments of the method, the CasX protein and the gNA are associated together in a protein complex, for example a ribonuclear protein complex (RNP).

In other embodiments, the methods of treating a disease in a subject in need thereof comprise administering to the subject a) a CasX protein or a polynucleotide encoding a CasX protein, b) a guide nucleic acid (gNA) comprising a targeting sequence or a polynucleotide encoding a gNA wherein the targeting sequence of the gNA has a sequence that hybridizes with the target nucleic acid, and c) a donor template comprising at least a portion or the entirety of a gene to be modified.

In some embodiments of the method of treating a disease, wherein a vector is administered to the subject, the vector is administered at a dose of at least about 1×10⁹ vector genomes (vg), at least about 1×10¹⁰ vg, at least about 1×10¹¹ vg, at least about 1×10¹² vg, at least about 1×10¹³ vg, at least about 1×10¹⁴ vg, at least about 1×10¹⁵ vg, or at least about 1×10¹⁶ vg. The vector can be administered by a route of administration selected from the group consisting of intraparenchymal, intravenous, intra-arterial, intracerebroventricular, intracisternal, intrathecal, intracranial, intravitreal, subretinal, and intraperitoneal routes.

A number of therapeutic strategies have been used to design the compositions for use in the methods of treatment of a subject with a disease. In some embodiments, the invention provides a method of treatment of a subject having a disease, the method comprising administering to the subject a CasX:gNA composition or a vector of any of the embodiments disclosed herein according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose. In exemplary embodiments the CasX:gNA composition comprises a CasX variant of any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, or a vector encoding the same. In some embodiments of the treatment regimen, the therapeutically effective dose of the composition or vector is administered as a single dose. In other embodiments of the treatment regimen, the therapeutically effective dose is administered to the subject as two or more doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In some embodiments of the treatment regiment, the effective doses are administered by a route selected from the group consisting of subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intralumbar, intrathecal, subarachnoid, intraventricular, intracapsular, intravenous, intralymphatical, intravitreal, subretinal, or intraperitoneal routes, wherein the administering method is injection, transfusion, or implantation.

In some embodiments of the methods of treatment of a subject with a disease, the method comprises administering to the subject a CasX:gNA composition as an RNP within a VLP disclosed herein according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose.

In some embodiments, the administering of the therapeutically effective amount of a CasX:gNA modality, including a vector comprising a polynucleotide encoding a CasX protein and a guide nucleic acid, or the administering of a CasX-gNA composition disclosed herein, to knock down or knock out expression of a gene product to a subject with a disease leads to the prevention or amelioration of the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disease. In some embodiments, the administration of the therapeutically effective amount of the CasX-gNA modality leads to an improvement in at least one clinically-relevant parameter for a disease.

In embodiments in which two or more different targeting complexes are provided to the cell (e.g., two gNA comprising two or more different spacers that are complementary to different sequences within the same or different target nucleic acid), the complexes may be provided simultaneously (e.g. as two polypeptides and/or nucleic acids), or delivered simultaneously. Alternatively, they may be provided consecutively. e.g. the targeting complex being provided first, followed by the second targeting complex, etc. or vice versa.

To improve the delivery of a DNA vector into a target cell, the DNA can be protected from damage and its entry into the cell facilitated, for example, by using lipoplexes and polyplexes. Thus, in some cases, a nucleic acid of the present disclosure (e.g., a recombinant expression vector of the present disclosure) can be covered with lipids in an organized structure like a micelle, a liposome, or a lipid nanoparticle. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids, anionic (negatively-charged), neutral, or cationic (positively-charged). Lipoplexes that utilize cationic lipids have proven utility for gene transfer. Cationic lipids, due to their positive charge, naturally complex with the negatively charged DNA. Also as a result of their charge, they interact with the cell membrane. Endocytosis of the lipoplex then occurs, and the DNA is released into the cytoplasm. The cationic lipids also protect against degradation of the DNA by the cell.

Complexes of polymers with DNA are referred to as polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis) such as inactivated adenovirus must occur. However, this is not always the case; polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

Dendrimers, a highly branched macromolecule with a spherical shape, may be also be used to genetically modify stem cells. The surface of the dendrimer particle may be functionalized to alter its properties. In particular, it is possible to construct a cationic dendrimer (i.e., one with a positive surface charge). When in the presence of genetic material such as a DNA plasmid, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination, the dendrimer-nucleic acid complex can be taken up into a cell by endocytosis.

In some cases, a nucleic acid of the disclosure (e.g., an expression vector) includes an insertion site for a guide sequence of interest. For example, a nucleic acid can include an insertion site for a guide sequence of interest, where the insertion site is immediately adjacent to a nucleotide sequence encoding the portion of a gNA variant (e.g. the scaffold region) that does not change when the guide sequence is changed to hybridize to a desired target sequence. Thus, in some cases, an expression vector includes a nucleotide sequence encoding a gNA, except that the portion encoding the spacer sequence portion of the gNA is an insertion sequence (an insertion site). An insertion site is any nucleotide sequence used for the insertion of a spacer in the desired sequence. “Insertion sites” for use with various technologies are known to those of ordinary skill in the art and any convenient insertion site can be used. An insertion site can be for any method for manipulating nucleic acid sequences. For example, in some cases the insertion site is a multiple cloning site (MCS) (e.g., a site including one or more restriction enzyme recognition sequences), a site for ligation independent cloning, a site for recombination based cloning (e.g., recombination based on att sites), a nucleotide sequence recognized by a CRISPR/Cas (e.g. Cas9) based technology, and the like.

IX. Cells

In still further embodiments, provided herein are cells comprising components of any of the CasX:gNA systems described herein. In some embodiments, the cells comprise any of the gNA variant embodiments as described herein, or the reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4 and further comprises a spacer that is complementary to the target DNA. In some embodiments, the cells further comprise a CasX variant as described herein (e.g, the sequences of Tables 3, 8, 9, 10 and 12 or a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO. 3). In other embodiments, the cells comprise RNP of any of the CasX:gNA embodiments described herein. In other embodiments, the disclosure provides cells comprising vectors encoding the CasX:gNA systems of any of the embodiments described herein. In still other embodiments, the cells comprise target DNA that has been edited by the CasX:gNA embodiments described herein; either to correct a mutation (knock-in) or to knock-down or knock-out a defective gene.

In some embodiments, the cell is a eukaryotic cell, for example a human cell. In alternative embodiments, the cell is a prokaryotic cell.

In some embodiments, the cell is a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding a CasX variant protein of the disclosure. In some embodiments, the genetically modified cell is genetically modified with an mRNA comprising a nucleotide sequence encoding a CasX variant protein. In some embodiments, the cell is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding a CasX variant protein of the present disclosure; and b) a nucleotide sequence encoding a gNA of the disclosure, and, optionally, comprises a nucleotide sequence encoding a donor template. In some cases, such cells are used to produce the individual components or RNP of CasX:gNA systems for use in editing target DNA. In other cases, cells that have been genetically modified in this way may be administered to a subject for purposes such as gene therapy, e.g., to treat a disease or condition caused by a genetic mutation or defect.

A cell that can serve as a recipient for a CasX variant protein and/or gNA of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a CasX variant protein and/or a gNA variant, can be any of a variety of cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cells of an immortalized cell line; cancer cells; animal cells; plant cells; algal cells; fungal cells; etc. A cell can be a recipient of a CasX RNP of the present disclosure. A cell can be a recipient of a single component of a CasX system of the present disclosure. A cell can be a recipient of a vector encoding the CasX, gNA and, optionally, a donor template of the CasX:gNA systems of any of the embodiments described herein.

Non-limiting examples of cells that can serve as host cells for production of the CasX:gNA systems disclosed herein include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

In certain embodiments, as provided herein, a cell can be an in vitro cell (e.g., established cultured cell line including, but not limited to HEK293 cells, HEK293T cells, HEK293-F cells, Lenti-X 293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, A549 cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS, WI38 cells, MRC5 cells, HeLa, HT1080 cells, or CHO cells). A cell can be an ex vivo cell (cultured cell from an individual). Such cells can be autologous with respect to a subject to be administered said cell(s). In other embodiments, the cells can be allogeneic with respect to a subject to be administered said cell(s). A cell can be an in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.

Suitable cells may include, in some embodiments, a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplated expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow-cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, fibroblasts, osteoblasts, chondrocytes, exogenous cell, endogenous cell, stem cell, hematopoietic stem cell, bone-marrow derived progenitor cell, myocardial cell, skeletal cell, fetal cell, undifferentiated cell, multi-potent progenitor cell, unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, and a post-natal stem cell.

In some embodiments, the cell is an immune cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg). In some cases, the cell expresses a chimeric antigen receptor.

In some embodiments, the cell is a stem cell. Stem cells may include, for example, adult stem cells. Adult stem cells can also be referred to as somatic stem cells. In some embodiments, the stem cell is a hematopoietic stem cell (HSC), neural stem cell or a mesenchymal stem cell. In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC.

A cell in some embodiments is an arthropod cell.

X. Kits and Articles of Manufacture

In another aspect, provided herein are kits comprising a CasX protein and one or a plurality of gNA of any of the embodiments of the disclosure and a suitable container (for example a tube, vial or plate). In some embodiments, the kit comprises a gNA variant of the disclosure, or the reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4. Exemplary gNA variants that can be included comprise a sequence of any one of SEQ ID NO: 2101-2280.

In some embodiments, the kit comprises a CasX variant protein of the disclosure (e.g. a sequence of Tables 3, 8, 9, 10 and 12), or the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In exemplary embodiments, a kit of the disclosure comprises a CasX variant of any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, the kit comprises a CasX variant of any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 44124415. In some embodiments, the kit comprises a CasX variant of any one of 3498-3501, 3505-3520, and 3540-3549.

In some embodiments, the kit comprises a gNA or a vector encoding a gNA, wherein the gNA comprises a sequence selected from the group consisting of SEQ ID NOS: 412-3295. In some embodiments, the gNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2101-2280. In some embodiments, the gNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280.

In certain embodiments, provided herein are kits comprising a CasX protein and gNA editing pair comprising a CasX variant protein of Tables 3, 8, 9, 10 and 12 and a gNA variant as described herein (e.g., a sequence of Table 2). In exemplary embodiments, a kit of the disclosure comprises a CasX and gNA editing pair, wherein the CasX variant comprises of any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 44124415. In some embodiments, the gNA of the gene editing pair comprises any one of SEQ ID NOS: 412-3295. In some embodiments, the gNA of the gene editing pair comprises any one of SEQ ID NOS: 2101-2280. In some embodiments, the gNA of the gene editing pair comprises any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

In some embodiments, the kit further comprises a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing. In some embodiments, the kit further comprises a pharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, the kit comprises appropriate control compositions for gene editing applications, and instructions for use.

In some embodiments, the kit comprises a vector comprising a sequence encoding a CasX variant protein of the disclosure, a gNA variant of the disclosure, optionally a donor template, or a combination thereof.

The present description sets forth numerous exemplary configurations, methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments. Embodiments of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below:

EMBODIMENT SET #1

Embodiment 1. A variant of a reference CasX protein, wherein the CasX variant is capable of forming a complex with a guide nucleic acid, and wherein the complex binds a target nucleic acid, and wherein the CasX variant comprises at least one modification in at least one of the following domains of the reference CasX protein:

(a) a non-target strand binding (NTSB) domain that binds to the non-target strand of DNA, wherein the NTSB domain comprises a four-stranded beta sheet;

(b) a target strand loading (TSL) domain that places the target DNA in a cleavage site of the CasX variant, the TSL domain comprising three positively charged amino acids, wherein the three positively charged amino acids bind to the target strand of DNA,

(c) a helical I domain that interacts with both the target DNA and a spacer region of a guide RNA, wherein the helical I domain comprises one or more alpha helices;

(d) a helical II domain that interacts with both the target DNA and a scaffold stem of the guide RNA;

(e) an oligonucleotide binding domain (OBD) that binds a triplex region of the guide RNA; and

(f) a RuvC DNA cleavage domain;

wherein the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein.

Embodiment 2. The CasX variant of Embodiment 1, wherein the reference CasX comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or at least 60% similarity thereto.

Embodiment 3. The CasX variant of Embodiment 2, wherein the reference CasX comprises the sequence of SEQ ID NO: 1, or at least 60% similarity thereto.

Embodiment 4. The CasX variant of Embodiment 2, wherein the reference CasX comprises the sequence of SEQ ID NO: 2, or at least 60% similarity thereto.

Embodiment 5. The CasX variant of Embodiment 2, wherein the reference CasX comprises the sequence of SEQ ID NO: 3, or at least 60% similarity thereto.

Embodiment 6. The CasX variant of any one of Embodiment 1 to Embodiment 5, wherein the complex binds a target DNA and cleaves the target DNA.

Embodiment 7. The CasX variant of any one of Embodiment 1 to Embodiment 5, wherein the complex binds a target DNA but does not cleave the target DNA.

Embodiment 8. The CasX variant of any one of Embodiment 1 to Embodiment 5, wherein the complex binds a target DNA and generates a single stranded nick in the target DNA.

Embodiment 9. The CasX variant of any one of Embodiment 1 to Embodiment 8, wherein at least one modification comprises at least one amino acid substitution in a domain.

Embodiment 10. The CasX variant of any one of Embodiment 1 to Embodiment 9, wherein at least one modification comprises at least one amino acid deletion in a domain.

Embodiment 11. The CasX variant of Embodiment 10, wherein at least one modification comprises the deletion of 1 to 4 consecutive or non-consecutive amino acids in the protein.

Embodiment 12. The CasX variant of any one of Embodiment 1 to Embodiment 10, wherein modification comprises at least one amino acid insertion in a domain.

Embodiment 13. The CasX variant of Embodiment 12, wherein at least one modification comprises the insertion of 1 to 4 consecutive or non-consecutive amino acids in a domain.

Embodiment 14. The CasX variant of any one of 1 to Embodiment 13, having at least 60% similarity to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 15. The CasX variant of Embodiment 14, wherein the variant has at least 60% similarity sequence identity to SEQ ID NO: 2.

Embodiment 16. The CasX variant of any one of Embodiment 1 to Embodiment 15, wherein the improved characteristic is selected from the group consisting of improved folding of the variant, improved binding affinity to the guide RNA, improved binding affinity to the target DNA, altered binding affinity to one or more PAM sequences, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved protein:guide RNA complex stability, improved protein solubility, improved protein:guide RNA complex solubility, improved protein yield, and improved fusion characteristics.

Embodiment 17. The CasX variant of any one of Embodiment 1 to Embodiment 16, wherein at least one of the at least one improved characteristic of the CasX variant is at least about 1.1 to about 100,000 times improved relative to the reference protein.

Embodiment 18. The CasX variant of any one of Embodiment 1 to Embodiment 17, wherein at least one of the at least one improved characteristics of the CasX variant is at least about 10 to about 100 times improved relative to the reference protein.

Embodiment 19. The CasX variant any one of Embodiment 1 to Embodiment 18, wherein the CasX variant has about 1.1 to about 100 times increased binding affinity to the guide RNA compared to the protein of SEQ ID NO: 2.

Embodiment 20. The CasX variant any one of Embodiment 1 to Embodiment 19, wherein the CasX variant has about one to about two times increased binding affinity to the target DNA compared to the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 21. The CasX variant of any one of Embodiment 1 to Embodiment 20, wherein the CasX protein comprises between 400 and 3000 amino acids.

Embodiment 22. The CasX variant of any one of Embodiment 1 to Embodiment 21, comprising at least one modification in at least two domains of the reference CasX protein.

Embodiment 23. The CasX variant of any one of Embodiment 1 to Embodiment 22, comprising two or more modifications in at least one domain of the reference CasX protein.

Embodiment 24. The CasX variant of any one of Embodiment 1 to Embodiment 23, wherein at least one modification comprises deletion of at least a portion of one domain of the reference CasX protein.

Embodiment 25. The CasX variant of any one of Embodiment 1 to Embodiment 24, comprising at least one modification of a region of non-contiguous residues that form a channel in which guide RNA:target DNA complexing occurs.

Embodiment 26. The CasX variant of any one of Embodiment 1 to Embodiment 25, comprising at least one modification of a region of non-contiguous residues that form an interface which binds with the guide RNA.

Embodiment 27. The CasX variant of any one of Embodiment 1 to Embodiment 26, comprising at least one modification of a region of non-contiguous residues that form a channel which binds with the non-target strand DNA.

Embodiment 28. The CasX variant of any one of Embodiment 1 to Embodiment 27, comprising at least one modification of a region of non-contiguous residues that form an interface which binds with the PAM.

Embodiment 29. The CasX variant of any one of Embodiment 1 to Embodiment 28, comprising at least one modification of a region of non-contiguous surface-exposed residues.

Embodiment 30. The CasX variant of any one of Embodiment 1 to Embodiment 29, comprising at least one modification of a region of non-contiguous residues that form a core through hydrophobic packing in a domain of the variant.

Embodiment 31. The CasX variant of any one of Embodiment 1 to Embodiment 30, wherein between 2 to 15 residues of the region are charged.

Embodiment 32. The CasX variant of any one of Embodiment 1 to Embodiment 31, wherein between 2 to 15 residues of the region are polar.

Embodiment 33. The CasX variant of any one of Embodiment 1 to Embodiment 32, wherein between 2 to 15 residues of the region stack with DNA or RNA bases.

Embodiment 34. A variant of a reference guide nucleic acid (NA) capable of binding a reference CasX protein, wherein:

the reference nucleic acid comprises a tracrNA sequence and a crNA sequence, wherein:

-   -   the tracrNA comprises a scaffold stem loop region comprising an         bubble,     -   the tracrNA and the crNA form a stem and a triplex region, and     -   the tracrNA and the crNA are fused, and form a fusion stem loop         region;

the variant comprises at least one modification to the reference guide NA, and

the variant exhibits at least one improved characteristic compared to the reference guide RNA.

Embodiment 35. The guide NA variant of Embodiment 34, comprising a tracrRNA stem loop comprising the sequence -UUU-N₃₋₂₀-UUU-.

Embodiment 36. The guide NA variant of Embodiment 34 or Embodiment 35, comprising a crRNA sequence with -AAAG- in a location 5′ to the spacer region.

Embodiment 37. The guide NA variant of Embodiment 36, wherein the -AAAG-sequence is immediately 5′ to the spacer region.

Embodiment 38. The guide NA variant of any one of Embodiment 34 to Embodiment 37, wherein the at least one improved characteristic is selected from the group consisting of improved stability, improved solubility, improved resistance to nuclease activity, increased folding rate of the NA, decreased side product formation during folding, increased productive folding, improved binding affinity to a reference CasX protein, improved binding affinity to a target DNA, improved gene editing, and improved specificity.

Embodiment 39. The guide NA variant of any one of Embodiment 34 to Embodiment 37, wherein at least one modification comprises at least one nucleic acid substitution in a region.

Embodiment 40. The guide NA variant of any one of Embodiment 34 to Embodiment 39, wherein at least one modification comprises at least one nucleic acid deletion in a region.

Embodiment 41. The guide NA variant of Embodiment 40, wherein at least one modification comprises deletion of 1 to 4 nucleic acids in a region.

Embodiment 42. The guide NA variant of any one of Embodiment 34 to Embodiment 40, wherein at least one modification comprises at least one nucleic acid insertion in a region.

Embodiment 43. The guide NA variant of Embodiment 42, wherein at least one modification comprises insertion of 1 to 4 nucleic acids in a region.

Embodiment 44. The guide NA variant of any one of Embodiment 34 to Embodiment 42, comprising a scaffold region at least 60% homologous to SEQ ID NO: 5.

Embodiment 45. The guide NA variant of any one of Embodiment 34 to Embodiment 44, comprising a scaffold NA stem loop at least 60% homologous to SEQ ID NO: 6.

Embodiment 46. The guide NA variant of any one of Embodiment 34 to Embodiment 45, comprising an extended stem loop at least 60% homologous to SEQ ID NO: 7.

Embodiment 47. The guide NA variant of any one of Embodiment 34 to Embodiment 46, wherein the guide NA variant sequence is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% homologous to SEQ ID NO: 4.

Embodiment 48. The guide NA variant of any one of Embodiment 34 to Embodiment 47, comprising an extended stem loop region comprising fewer than 10,000 nucleotides.

Embodiment 49. The guide NA variant of any one of Embodiment 34 to Embodiment 44, wherein the scaffold stem loop or the extended stem loop is swapped for an exogenous stem loop.

Embodiment 50. The guide NA variant of any one of Embodiment 34 to Embodiment 49, further comprising a hairpin loop that is capable of binding a protein, RNA or DNA.

Embodiment 51. The guide NA variant of Embodiment 50, wherein the hairpin loop is from MS2, QB, U1A, or PP7.

Embodiment 52. The guide NA variant of any one of Embodiment 34 to Embodiment 48, further comprising one or more ribozymes.

Embodiment 53. The guide NA variant of Embodiment 52, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA variant.

Embodiment 54. The guide NA variant of Embodiment 52 or Embodiment 53, wherein at least one of the one or more ribozymes are an hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

Embodiment 55. The guide NA variant of any one of Embodiment 34 to Embodiment 54, further comprising a protein binding motif.

Embodiment 56. The guide NA variant of any one of Embodiment 34 to Embodiment 55, further comprising a thermostable stem loop.

Embodiment 57. The guide NA variant of Embodiment 34, comprising the sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 66.

Embodiment 58. The guide NA variant of any one of Embodiment 34 to Embodiment 57, further comprising a spacer region.

Embodiment 59. The guide NA variant of any one of Embodiment 34 to Embodiment 58, wherein the reference guide RNA comprises SEQ ID NO: 5.

Embodiment 60. The guide NA variant of any one of Embodiment 38 to Embodiment 59, wherein the reference CasX protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 61. A gene editing pair comprising a CRISPR-associated protein (Cas protein) and a guide NA, wherein the Cas protein is a CasX variant of any one of Embodiment 1 to Embodiment 33.

Embodiment 62. The gene editing pair of 61, wherein the guide NA is a guide NA variant of any one of Embodiment 34 to Embodiment 60, or the guide NA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 63. The gene editing pair of Embodiment 61 or Embodiment 62, wherein the gene editing pair has one or more improved characteristics compared to a gene editing pair comprising a CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and a guide RNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 64. The gene editing pair of Embodiment 63, wherein the one or more improved characteristics comprises improved protein:guide NA complex stability, improved protein:guide NA complex stability, improved binding affinity between the protein and guide NA, improved kinetics of complex formation, improved binding affinity to the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity.

Embodiment 65. A gene editing pair comprising a CRISPR-associated protein (Cas protein) and a guide NA, wherein the guide NA is a guide NA variant of any one of Embodiment 34 to Embodiment 60.

Embodiment 66. The gene editing pair of Embodiment 65, wherein the Cas protein is a CasX variant of any one of Embodiment 1 to Embodiment 22, or a CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO. 3.

Embodiment 67. The gene editing pair of Embodiment 65 or Embodiment 66, wherein the gene editing pair has one or more improved characteristics.

Embodiment 68. The gene editing pair of Embodiment 67, wherein the one or more improved characteristics comprises improved protein:guide NA complex stability, improved protein:guide NA complex stability, improved binding affinity between the protein and guide NA, improved binding affinity to the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity.

Embodiment 69. A method of editing a target DNA, comprising combining the target DNA with a gene editing pair, the gene editing pair comprising a CasX variant and a guide RNA, wherein the CasX variant is a CasX variant of any one of Embodiment 1 to Embodiment 33, and wherein the combining results in editing of the target DNA.

Embodiment 70. The method of 69, wherein the guide NA is a guide NA variant of any one of Embodiment 34 to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 71. The method of Embodiment 69 or Embodiment 70, wherein editing occurs in vitro outside of a cell.

Embodiment 72. The method of Embodiment 69 or Embodiment 70, wherein editing occurs in vitro inside of a cell.

Embodiment 73. The method of Embodiment 69 or Embodiment 70, wherein editing occurs in vivo inside of a cell.

Embodiment 74. The method of any one of Embodiment 71 to Embodiment 73, wherein the cell is a eukaryotic cell.

Embodiment 75. The method of Embodiment 74, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a protist cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

Embodiment 76. The method of any one of Embodiment 71 to Embodiment 73, wherein the cell is a prokaryotic cell.

Embodiment 77. A method of editing a target DNA, comprising combining the target DNA with a gene editing pair, the gene editing pair comprising a CRISPR-associated protein (Cas protein) and a guide NA variant, wherein the guide NA variant is a guide NA variant of any one of Embodiment 34 to Embodiment 60, and wherein the combining results in editing of the target DNA.

Embodiment 78. The method of Embodiment 77, wherein the Cas protein is a CasX variant of any one of Embodiment 1 to Embodiment 33, or a CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 79. The method of Embodiment 77 or Embodiment 78, wherein editing occurs in vitro outside of a cell.

Embodiment 80. The method of Embodiment 77 or Embodiment 78, wherein editing occurs in vitro inside of a cell.

Embodiment 81. The method of Embodiment 77 or Embodiment 78, wherein contacting occurs in vivo inside of a cell.

Embodiment 82. The method of any one of Embodiment 79 to Embodiment 81, wherein the cell is a eukaryotic cell.

Embodiment 83. The method of Embodiment 82, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

Embodiment 84. The method of any one of Embodiment 79 to Embodiment 81, wherein the cell is a prokaryotic cell.

Embodiment 85. A cell comprising a CasX variant, wherein the CasX variant is a CasX variant of any one of Embodiment 1 to Embodiment 33.

Embodiment 86. The cell of Embodiment 85, further comprising a guide NA variant of any one of Embodiment 34 to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 87. A cell comprising a guide NA variant, wherein the guide NA variant is a guide NA variant of any one of Embodiment 34 to Embodiment 60.

Embodiment 88. The cell of Embodiment 87, further comprising a CasX variant of any one of Embodiment 1 to Embodiment 33, or a CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO. 3.

Embodiment 89. The cell of any one of 85 to Embodiment 88, wherein the cell is a eukaryotic cell.

Embodiment 90. The cell of any one of 85 to Embodiment 88, wherein the cell is a prokaryotic cell.

Embodiment 91. A polynucleotide encoding the CasX variant of any one of Embodiment 1 to Embodiment 33.

Embodiment 92. A vector comprising the polynucleotide of Embodiment 91.

Embodiment 93. The vector of Embodiment 92, wherein the vector is a bacterial plasmid.

Embodiment 94. A cell comprising the polynucleotide of Embodiment 91, or the vector of Embodiment 92 or Embodiment 93.

Embodiment 95. A composition, comprising the CasX variant of any one of Embodiment 1 to Embodiment 33.

Embodiment 96. The composition of 95, further comprising a guide RNA variant of any one of Embodiment 34 to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 97. The composition of Embodiment 95 or Embodiment 96, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 98. A composition, comprising a guide RNA variant of any one of Embodiment 34 to Embodiment 60.

Embodiment 99. The composition of Embodiment 98, further comprising the CasX variant of any one of 1 to Embodiment 33, or the CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 100. The composition of Embodiment 98 or Embodiment 99, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 101. A composition, comprising the gene editing pair of any one of Embodiment 61 to Embodiment 68.

Embodiment 102. The composition of Embodiment 101, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 103. A kit, comprising the CasX variant of any one of Embodiment 1 to Embodiment 33 and a container.

Embodiment 104. The kit of Embodiment 103, further comprising a guide NA variant of any one of Embodiment 34 to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 105. The kit of Embodiment 103 or Embodiment 104, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 106. A kit, comprising a guide NA variant of any one of Embodiment 34 to Embodiment 60.

Embodiment 107. The kit of 106, further comprising the CasX variant of any one of Embodiment 1 to Embodiment 33, or the CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 108. The kit of Embodiment 106 or Embodiment 107, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 109. A kit, comprising the gene editing pair of any one of Embodiment 61 to Embodiment 68.

Embodiment 110. The kit of Embodiment 109, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 111. A CasX variant comprising any one of the sequences listed in Table 3.

Embodiment 112. A guide RNA variant comprising any one of the sequences listed in Table 1 or Table 2.

Embodiment 113. The CasX variant of any one of Embodiment 1 to Embodiment 33, wherein the reference CasX protein comprises a first domain from a first CasX protein and second domain from a second CasX protein.

Embodiment 114. The CasX variant of Embodiment 113, wherein the first domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

Embodiment 115. The CasX variant of Embodiment 113, wherein the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

Embodiment 116. The method of any one of Embodiment 113 to Embodiment 115, wherein the first and second domains are not the same domain.

Embodiment 117. The CasX variant of any one of Embodiment 113 to Embodiment 116, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 2.

Embodiment 118. The CasX variant of any one of Embodiment 113 to Embodiment 116, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 119. The CasX variant of any one of Embodiment 113 to Embodiment 116, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 120. The CasX variant of any one of Embodiment 1 to Embodiment 33 or 113 to Embodiment 119, wherein the CasX protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second CasX protein.

Embodiment 121. The CasX variant of Embodiment 120, wherein the at least one chimeric domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

Embodiment 122. The CasX variant of Embodiment 120 or Embodiment 121, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 2.

Embodiment 123. The CasX variant of Embodiment 120 or Embodiment 121, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 124. The CasX variant of Embodiment 120 or Embodiment 121, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 125. The CasX variant of Embodiment 120, wherein the at least one chimeric comprises a chimeric RuvC domain.

Embodiment 126. The CasX variant of 125, wherein the chimeric RuvC domain comprises amino acids 661 to Embodiment 824 of SEQ ID NO: 1 and amino acids 922 to Embodiment 978 of SEQ ID NO: 2.

Embodiment 127. The CasX variant of 125, wherein the chimeric RuvC domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ ID NO: 1.

Embodiment 128. The guide NA variant of any one of 34 to Embodiment 60, wherein the reference guide NA comprises a first region from a first guide NA and a second region from a second guide NA.

Embodiment 129. The guide NA variant of 128, wherein the first region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

Embodiment 130. The guide NA variant of 128 or 129, wherein the second region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

Embodiment 131. The guide NA variant of any one of Embodiments 128 to Embodiment 130, wherein the first and second regions are not the same region.

Embodiment 132. The guide NA variant of any one of Embodiments 128 to Embodiment 131, wherein the first guide NA comprises a sequence of SEQ ID NO: 4 and the second guide NA comprises a sequence of SEQ ID NO: 5.

Embodiment 133. The guide NA variant of any one of Embodiments 34-60 or Embodiments 128-132, comprising at least one chimeric region comprising a first part from a first guide NA and a second part from a second guide NA.

Embodiment 134. The guide NA variant of Embodiment 133, wherein the at least one chimeric region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

Embodiment 135. The guide NA variant of Embodiment 134, wherein the first guide NA comprises a sequence of SEQ ID NO: 4 and the second guide NA comprises a sequence of SEQ ID NO: 5.

EMBODIMENT SET #2

Embodiment 1. A variant of a reference CasX protein, wherein the CasX variant is capable of forming a complex with a guide nucleic acid (gNA), and wherein the complex can bind a target nucleic acid, and wherein the CasX variant comprises at least one modification in at least one domain of the reference CasX protein selected from:

a. a non-target strand binding (NTSB) domain that binds to the non-target strand of DNA, wherein the NTSB domain comprises a four-stranded beta sheet;

b. a target strand loading (TSL) domain that places the target DNA in a cleavage site of the CasX variant, the TSL domain comprising three positively charged amino acids, wherein the three positively charged amino acids bind to the target strand of DNA,

c. a helical I domain that interacts with both the target DNA and a targeting sequence of a gNA, wherein the helical I domain comprises one or more alpha helices;

d. a helical II domain that interacts with both the target DNA and a scaffold stem of the gNA;

e. an oligonucleotide binding domain (OBD) that binds a triplex region of the gNA; or

f. a RuvC DNA cleavage domain;

wherein the CasX variant exhibits one or more improved characteristics as compared to the reference CasX protein.

Embodiment 2. The CasX variant of Embodiment 1, wherein the CasX reference comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 3. The CasX variant of Embodiment 1 or Embodiment 2, wherein the at least one modification comprises at least one amino acid substitution in a domain of the CasX variant.

Embodiment 4. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the substitution of 1 to 10 consecutive or non-consecutive amino acid substitutions in the CasX variant.

Embodiment 5. The CasX variant of any one of the preceding Embodiments, wherein at least one modification comprises at least one amino acid deletion in a domain of the CasX variant.

Embodiment 6. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the deletion of 1 to 10 consecutive or non-consecutive amino acids in the CasX variant.

Embodiment 7. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the substitution of 1 to 10 consecutive or non-consecutive amino acid substitutions and the deletion of 1 to 10 consecutive or non-consecutive amino acids in the CasX variant.

Embodiment 8. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises at least one amino acid insertion in a domain of the CasX variant.

Embodiment 9. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the insertion of 1 to 4 consecutive or non-consecutive amino acids in a domain of the CasX variant.

Embodiment 10. The CasX variant of any one of the preceding Embodiments, wherein the CasX variant has a sequence selected from the group consisting of the sequences of Table 3, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity thereto.

Embodiment 11. The CasX variant of any one of the preceding Embodiments, wherein the CasX protein has binding affinity for a protospacer adjacent motif (PAM) sequence selected from the group consisting of TTC, ATC, GTC, and CTC.

Embodiment 12. The CasX variant of any one of the preceding Embodiments, wherein the CasX protein further comprises one or more nuclear localization signals (NLS).

Embodiment 13. The CasX variant of Embodiment 12, wherein the one or more NLS are selected from the group of sequences consisting of PKKKRKV (SEQ ID NO: 352), KRPAATKKAGQAKKKK (SEQ ID NO: 353), PAAKRVKLD (SEQ ID NO: 354), RQRRNELKRSP (SEQ ID NO: 355), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 356), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 357), VSRKRPRP (SEQ ID NO: 358), PPKKARED (SEQ ID NO: 359), PQPKKKPL (SEQ ID NO: 360), SALIKKKKKMAP (SEQ ID NO: 361), DRLRR (SEQ ID NO: 362), PKQKKRK (SEQ ID NO: 363), RKLKKKIKKL (SEQ ID NO: 364), REKKKFLKRR (SEQ ID NO: 365), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 366), RKCLQAGMNLEARKTKK (SEQ ID NO: 367), PRPRKIPR (SEQ ID NO: 368), PPRKKRTVV (SEQ ID NO: 369), NLSKKKKRKREK (SEQ ID NO: 370), RRPSRPFRKP (SEQ ID NO: 371), KRPRSPSS (SEQ ID NO: 372), KRGINDRNFWRGENERKTR (SEQ ID NO: 373), PRPPKMARYDN (SEQ ID NO: 374), KRSFSKAF (SEQ ID NO: 375), KLKIKRPVK (SEQ ID NO: 376), PKTRRRPRRSQRKRPPT (SEQ ID NO: 378), RRKKRRPRRKKRR (SEQ ID NO: 381), PKKKSRKPKKKSRK (SEQ ID NO: 382), HKKKHPDASVNFSEFSK (SEQ ID NO: 383), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 384), LSPSLSPLLSPSLSPL (SEQ ID NO: 385), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 386), PKRGRGRPKRGRGR (SEQ ID NO: 387), and MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 411).

Embodiment 14. The CasX variant of Embodiment 12 or Embodiment 13, wherein the one or more NLS are expressed at the C-terminus of the CasX protein.

Embodiment 15. The CasX variant of Embodiment 12 or Embodiment 13, wherein the one or more NLS are expressed at the N-terminus of the CasX protein.

Embodiment 16. The CasX variant of Embodiment 12 or Embodiment 13, wherein the one or more NLS are expressed at the N-terminus and C-terminus of the CasX protein.

Embodiment 17. The CasX variant of any one of the preceding Embodiments, wherein the improved characteristic is selected from the group consisting of improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target DNA, altered binding affinity to one or more PAM sequences of the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target DNA strand, improved protein stability, improved protein:gNA complex stability, improved protein solubility, improved protein:gNA complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics.

Embodiment 18. The CasX variant of any one of the preceding Embodiments, wherein at least one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 19. The CasX variant of any one of the preceding Embodiments, wherein one or more of the improved characteristics of the CasX variant is at least about 10 to about 100-fold improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 20. The CasX variant any one of the preceding Embodiments, wherein the CasX variant has about 1.1 to about 100-fold increased binding affinity to the gNA compared to the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 21. The CasX variant any one of the preceding Embodiments, wherein the CasX variant has about 1.1 to about 10-fold increased binding affinity to the target DNA compared to the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 22. The CasX variant of any one of the preceding Embodiments, wherein the CasX variant comprises between 400 and 3000 amino acids.

Embodiment 23. The CasX variant of any one of the preceding Embodiments, comprising at least one modification in at least two domains of the CasX variant relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 24. The CasX variant of any one of the preceding Embodiments, comprising two or more modifications in at least one domain of the CasX variant relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 25. The CasX variant of any one of the preceding Embodiments, wherein at least one modification comprises deletion of at least a portion of one domain of the CasX variant relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 26. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel in which gNA:target DNA complexing with the CasX variant occurs.

Embodiment 27. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the gNA.

Embodiment 28. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel which binds with the non-target strand DNA.

Embodiment 29. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the PAM.

Embodiment 30. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous surface-exposed amino acid residues of the CasX variant.

Embodiment 31. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues that form a core through hydrophobic packing in a domain of the CasX variant.

Embodiment 32. The CasX variant of any one of Embodiments 25-30, wherein the modification is a deletion, an insertion, and/or a substitution of one or more amino acids of the region.

Embodiment 33. The CasX variant of any one of Embodiments 25-32, wherein between 2 to 15 amino acid residues of the region of the CasX variant are substituted with charged amino acids.

Embodiment 34. The CasX variant of any one of Embodiments 25-32, wherein between 2 to 15 amino acid residues of a region of the CasX variant are substituted with polar amino acids.

Embodiment 35. The CasX variant of any one of Embodiments 25-32, wherein between 2 to 15 amino acid residues of a region of the CasX variant are substituted with amino acids that stack with DNA or RNA bases.

Embodiment 36. The CasX variant of any one of the preceding Embodiments, wherein the CasX variant protein comprises a nuclease domain having nickase activity.

Embodiment 37. The CasX variant of any one of Embodiments 1-35, wherein the CasX variant protein comprises a nuclease domain having double-stranded cleavage activity.

Embodiment 38. The CasX variant of any one of Embodiments 1-35, wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the gNA retain the ability to bind to the target nucleic acid.

Embodiment 39. The CasX variant of Embodiment 38, wherein the dCasX comprises a mutation at residues:

a. D672, E769, and/or D935 corresponding to the CasX protein of SEQ ID NO: 1 or

b. D659, E756 and/or D922 corresponding to the CasX protein of SEQ ID NO: 2.

Embodiment 40. The CasX variant of Embodiment 39, wherein the mutation is a substitution of alanine for the residue.

Embodiment 41. A variant of a reference guide nucleic acid (gNA) capable of binding a CasX protein, wherein the reference guide nucleic acid comprises a tracrNA sequence and a crNA sequence, wherein:

a. the tracrNA comprises a scaffold stem loop region comprising a bubble;

b. the tracrNA and the crNA form a stem and a triplex region; and

c. the tracrNA and the crNA are fused, and form a fusion stem loop region

wherein the gNA variant comprises at least one modification compared to the reference guide nucleic acid sequence, and the variant exhibits one or more improved characteristics compared to the reference guide RNA.

Embodiment 42. The gNA variant of Embodiment 41, comprising a tracrRNA stem loop comprising the sequence -UUU-N₃₋₂₀-UUU- (SEQ ID NO: 4403).

Embodiment 43. The gNA variant of Embodiment 41 or 42, comprising a crRNA sequence with -AAAG- in a location 5′ to a targeting sequence of the gNA variant.

Embodiment 44. The gNA variant of Embodiment 43, wherein the -AAAG- sequence is immediately 5′ to the targeting sequence.

Embodiment 45. The gNA variant of any one of Embodiments 41-44, wherein the gNA variant further comprises a targeting sequence wherein the targeting sequence is complementary to the target DNA sequence.

Embodiment 46. The gNA variant of any one of Embodiments 41-45, wherein the one or more improved characteristics is selected from the group consisting of improved stability, improved solubility, improved resistance to nuclease activity, increased folding rate of the NA, decreased side product formation during folding, increased productive folding, improved binding affinity to a CasX protein, improved binding affinity to a target DNA, improved gene editing, and improved specificity.

Embodiment 47. The gNA variant of Embodiment 46, wherein the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 48. The CasX variant of Embodiment 46 or 47, wherein one or more of the improved characteristics of the CasX variant is at least about 10 to about 100-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 49. The gNA variant of any one of Embodiments 41-48, wherein the at least one modification comprises at least one nucleotide substitution in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 50. The gNA variant of Embodiment 41-49, wherein the at least one modification comprises substitution of at least 1 to 4 nucleotides in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 51. The gNA variant of any one of Embodiments 41-50, wherein the at least one modification comprises at least one nucleotide deletion in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 52. The gNA variant of Embodiments 41-51, wherein the at least one modification comprises deletion of 1 to 4 nucleotides in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 53. The gNA variant of any one of Embodiments 41-52, wherein the at least one modification comprises at least one nucleotide insertion in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 54. The gNA variant of any one of Embodiments 41-53, wherein the at least one modification comprises insertion of 1 to 4 nucleotides in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 55. The gNA variant of any one of Embodiments 41-54, wherein the at least one modification comprises a deletion of at least 1 to 4 nucleotides, an insertion of at least 1 to 4 nucleotides, a substitution of at least 1 to 4 nucleotides, or any combination thereof in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 56. The gNA variant of any one of Embodiments 41-5, comprising a scaffold region at least 60% homologous to SEQ ID NO: 4 or SEQ ID NO: 5.

Embodiment 57. The gNA variant of any one of Embodiments 41-55, comprising a scaffold NA stem loop at least 60% homologous to SEQ ID NO: 14.

Embodiment 58. The gNA variant of any one of Embodiments 41-55, comprising an extended stem loop at least 60% homologous to SEQ ID NO: 14.

Embodiment 59. The gNA variant of any one of Embodiments 41-55, wherein the gNA variant sequence is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, or at least 80% homologous to SEQ ID NO: 4.

Embodiment 60. The gNA variant of any one of Embodiments 41-58, wherein the gNA variant sequence is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, or is 100% homologous to a sequence selected from the group of sequences of SEQ ID NOS: 2101-2241.

Embodiment 61. The gNA variant of any one of Embodiments 41-60, comprising an extended stem loop region comprising fewer than 10,000 nucleotides.

Embodiment 62. The gNA variant of any one of Embodiments 41-60, wherein the scaffold stem loop or the extended stem loop sequence is replaced with an exogenous stem loop sequence.

Embodiment 63. The gNA variant of Embodiment t 62, wherein the exogenous stem loop is a hairpin loop that is capable of binding a protein, RNA or DNA molecule.

Embodiment 64. The gNA variant of Embodiment 62 or 63, wherein the exogenous stem loop is a hairpin loop that increases the stability of the gNA.

Embodiment 65. The gNA variant of Embodiment 63 or 64, wherein the hairpin loop is selected from MS2, Qβ, U1A, or PP7.

Embodiment 66. The gNA variant of any one of Embodiments 41-65, further comprising one or more ribozymes.

Embodiment 67. The gNA variant of Embodiment 66, wherein the one or more ribozymes are independently fused to a terminus of the gNA variant.

Embodiment 68. The gNA variant of Embodiment 66 or 67, wherein at least one of the one or more ribozymes are an hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

Embodiment 69. The gNA variant of any one of Embodiments 41-68, further comprising a protein binding motif.

Embodiment 70. The gNA variant of any one of Embodiments 41-69, further comprising a thermostable stem loop.

Embodiment 71. The gNA variant of Embodiment 41, comprising the sequence of any one of SEQ ID NO: 2101-2241.

Embodiment 72. The gNA variant of any one of Embodiments 41-71, further comprising a targeting sequence.

Embodiment 73. The gNA variant of Embodiment 72, wherein the targeting sequence has 14, 15, 16, 18, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.

Embodiment 74. The gNA variant of any one of Embodiments 41-73, wherein the gNA is chemically modified.

Embodiment 75. A gene editing pair comprising a CasX protein and a first gNA.

Embodiment 76. The gene editing pair of Embodiment 74, wherein the first gNA comprises:

a. a gNA variant of any one of Embodiments 41-74 and a targeting sequence; or

b. a reference guide nucleic acid of SEQ ID NOS: 4 or 5 and a targeting sequence,

wherein the targeting sequence is complementary to the target nucleic acid.

Embodiment 77. The gene editing pair of Embodiment 74 or 76, wherein the CasX comprises:

a. a CasX variant of any one of Embodiments 1-40; or

b. a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 78. The gene editing pair of any one of Embodiments 74-77, further comprising a second gNA or a nucleic acid encoding the second gNA, wherein the second gNA has a targeting sequence complementary to a different portion of the target nucleic acid compared to the targeting sequence of the first gNA.

Embodiment 79. The gene editing pair of any one of Embodiments 74-78, wherein the CasX protein and the gNA are capable of associating together in a ribonuclear protein complex (RNP).

Embodiment 80. The gene editing pair of any one of Embodiments 74-79, wherein the CasX protein and the gNA are associated together in a ribonuclear protein complex (RNP).

Embodiment 81. The gene editing pair of Embodiment 79 or 80, wherein the RNP is capable of binding a target DNA.

Embodiment 82. The gene editing pair of any one of Embodiments 79-81, wherein the RNP has a higher percentage of cleavage-competent RNP compared to an RNP of a reference CasX protein and a reference guide nucleic acid.

Embodiment 83. The gene editing pair of any one of Embodiments 79-82, wherein the RNP is capable of binding and cleaving a target DNA.

Embodiment 84. The gene editing pair of any one of Embodiments 79-82, wherein the RNP binds a target DNA but does not cleave the target DNA.

Embodiment 85. The gene editing pair of any one of Embodiments 79-83, wherein the RNP is capable of binding a target DNA and generating one or more single-stranded nicks in the target DNA.

Embodiment 86. The gene editing pair of any one of Embodiments 79-83 or 85, wherein the gene editing pair has one or more improved characteristics compared to a gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and a reference guide nucleic acid of SEQ ID NOS: 4 or 5.

Embodiment 87. The gene editing pair of Embodiment 86, wherein the one or more improved characteristics comprises improved CasX:gNA RNP complex stability, improved binding affinity between the CasX and gNA, improved kinetics of RNP complex formation, higher percentage of cleavage-competent RNP, improved RNP binding affinity to the target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity.

Embodiment 88. The gene editing pair of Embodiment 86 or 87, wherein the at least one or more of the improved characteristics is at least about 1.1 to about 100,000-fold improved relative to a gene editing pair of the reference CasX protein and the reference guide nucleic acid.

Embodiment 89. The gene editing pair of any one of Embodiments 86-88, wherein one or more of the improved characteristics of the CasX variant is at least about 10 to about 100-fold improved relative to a gene editing pair of the reference CasX protein and the reference guide nucleic acid.

Embodiment 90. A method of editing a target DNA, comprising contacting the target DNA with a gene editing pair of any one of Embodiments 74-89, wherein the contacting results in editing of the target DNA.

Embodiment 91. The method of Embodiment 90, comprising contacting the target DNA with a plurality of gNAs comprising targeting sequences complementary to different regions of the target DNA.

Embodiment 92. The method of Embodiment 90 or 91, wherein the contacting introduces one or more single-stranded breaks in the target DNA and wherein the editing comprises a mutation, an insertion, or a deletion in the target DNA.

Embodiment 93. The method of Embodiment 90 or 91, wherein the contacting comprises introducing one or more double-stranded breaks in the target DNA and wherein the editing comprises a mutation, an insertion, or a deletion in the target DNA.

Embodiment 94. The method of any one of Embodiments 90-93, further comprising contacting the target DNA with a nucleotide sequence of a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to the target DNA.

Embodiment 95. The method of Embodiment 94, wherein the donor template is inserted in the target DNA at the break site by homology-directed repair.

Embodiment 96. The method of any one of Embodiments 90-95, wherein editing occurs in vitro outside of a cell.

Embodiment 97. The method of any one of Embodiments 90-95, wherein editing occurs in vitro inside of a cell.

Embodiment 98. The method of any one of Embodiments 90-95, wherein editing occurs in vivo inside of a cell.

Embodiment 99. The method of Embodiments 97 or 98, wherein the cell is a eukaryotic cell.

Embodiment 100. The method of Embodiment 99, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a protist cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

Embodiment 101. The method of Embodiment 99 or 100, wherein the method comprises contacting the eukaryotic cell with a vector encoding or comprising the CasX protein and the gNA, and optionally further comprising the donor template.

Embodiment 102. The method of Embodiment 101, wherein the vector is an Adeno-Associated Viral (AAV) vector.

Embodiment 103. The method of Embodiment 102, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, or AAVRh10.

Embodiment 104. The method of Embodiment 101, wherein the vector is a lentiviral vector.

Embodiment 105. The method of Embodiment 101, wherein the vector is a virus-like particle (VLP).

Embodiment 106. The method of any one of Embodiments 101-105, wherein the vector is administered to a subject at a therapeutically effective dose.

Embodiment 107. The method of Embodiment 105, wherein the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

Embodiment 108. The method of Embodiment 107, wherein the subject is a human.

Embodiment 109. The method of any one of Embodiments 106-108, wherein the vector is administered at a dose of at least about 1×10¹⁰ vector genomes (vg), or at least about 1×10¹¹ vg, or at least about 1×10¹² vg, or at least about 1×10¹³ vg, or at least about 1×10¹⁴ vg, or at least about 1×10¹⁵ vg, or at least about 1×10¹⁶ vg.

Embodiment 110. The method of any one of Embodiments 106-109, wherein the vector is administered by a route of administration selected from the group consisting of intraparenchymal, intravenous, intra-arterial, intracerebroventricular, intracisternal, intrathecal, intracranial, and intraperitoneal routes.

Embodiment 111. The method of Embodiment 97, wherein the cell is a prokaryotic cell.

Embodiment 112. A cell comprising a CasX variant, wherein the CasX variant is a CasX variant of any one of Embodiments 1-40.

Embodiment 113. The cell of Embodiment 112, further comprising

a. a gNA variant of any one of Embodiments 41-74, or

b. a reference guide nucleic acid of SEQ ID NOS: 4 or 5 and a targeting sequence.

Embodiment 114. A cell comprising a gNA variant of any one of Embodiments 41-74.

Embodiment 115. The cell of Embodiment 114, further comprising a CasX variant of any one of Embodiments 1 to Embodiment 35, or a CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO. 3.

Embodiment 116. The cell of Embodiment 114 or 115, further comprising a donor nucleotide template comprising a sequence that hybridizes with a target DNA.

Embodiment 117. The cell of Embodiment 116, wherein the donor template ranges in size from 10-10,000 nucleotides.

Embodiment 118. The cell of Embodiment 116 or 117, wherein the donor template is a single-stranded DNA template or a single stranded RNA template.

Embodiment 119. The method of Embodiment 116 or 117, wherein the donor template is a double-stranded DNA template.

Embodiment 120. The cell of any one of Embodiments 112-119, wherein the cell is a eukaryotic cell.

Embodiment 121. The cell of any one of Embodiments 112-119, wherein the cell is a prokaryotic cell.

Embodiment 122. A polynucleotide encoding the CasX variant of any one of Embodiments 1 to 40.

Embodiment 123. A polynucleotide encoding the gNA variant of any one of Embodiments 41-74.

Embodiment 124. A vector comprising the polynucleotide of Embodiment 122 and/or 123.

Embodiment 125. The vector of Embodiment 123, wherein the vector is an Adeno-Associated Viral (AAV) vector.

Embodiment 126. The method of Embodiment 125, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, or AAVRh10.

Embodiment 127. The vector of Embodiment 123, wherein the vector is a lentiviral vector.

Embodiment 128. The vector of Embodiment 124, wherein the vector is a virus-like particle (VLP).

Embodiment 129. A cell comprising the polynucleotide of Embodiment 122, or the vector of any one of Embodiments 124-128.

Embodiment 130. A composition, comprising the CasX variant of any one of Embodiments 1 to 35.

Embodiment 131. The composition of Embodiment 130, further comprising:

a. a gNA variant of any one of Embodiments 45-74, or

b. the reference guide RNA of SEQ ID NOS: 4 or 5 and a targeting sequence.

Embodiment 132. The composition of Embodiment 130 or 131, wherein the CasX protein and the gNA are associated together in a ribonuclear protein complex (RNP).

Embodiment 133. The composition of any one of Embodiments 130-132, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

Embodiment 134. The composition of any one of Embodiments 130-133, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 135. A composition, comprising a gNA variant of any one of Embodiments 41-74.

Embodiment 136. The composition of Embodiment 135, further comprising the CasX variant of any one of Embodiments 1 to 35, or the CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 137. The composition of Embodiment 136, wherein the CasX protein and the gNA are associated together in a ribonuclear protein complex (RNP).

Embodiment 138. The composition of any one of Embodiments 135-137, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

Embodiment 139. The composition of any one of Embodiments 135-138, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 140. A composition, comprising the gene editing pair of any one of Embodiments 4-89.

Embodiment 141. The composition of Embodiment 140, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

Embodiment 142. The composition of Embodiment 140 or 141, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 143. A kit, comprising the CasX variant of any one of Embodiments 1 to 35 and a container.

Embodiment 144. The kit of Embodiment 143, further comprising:

a. a gNA variant of any one of Embodiments 45-74, or

b. the reference guide RNA of SEQ ID NOS: 4 or 5 and a targeting sequence.

Embodiment 145. The kit of Embodiment 143 or 144, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target DNA.

Embodiment 146. The kit of any one of Embodiments 143-145, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 147. A kit, comprising a gNA variant of any one of Embodiments 45-74.

Embodiment 148. The kit of Embodiment 147, further comprising the CasX variant of any one of Embodiments 1 to 35, or the CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 149. The kit of Embodiment 147 or 148, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target DNA.

Embodiment 150. The kit of any one of Embodiments 147-149, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 151. A kit, comprising the gene editing pair of any one of Embodiments 74-89.

Embodiment 152. The kit of Embodiment 151, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

Embodiment 153. The kit of Embodiment 151 or 152, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

Embodiment 154. A CasX variant comprising any one of the sequences listed in Table 3.

Embodiment 155. A gNA variant comprising any one of the sequences listed in Table 2.

Embodiment 156. The gNA variant of Embodiment 155, further comprising a targeting sequence of at least 10 to 30 nucleotides complementary to a target DNA.

Embodiment 157. The gNA variant of Embodiment 156, wherein the targeting sequence has 20 nucleotides.

Embodiment 158. The gNA variant of Embodiment 156, wherein the targeting sequence has 19 nucleotides.

Embodiment 159. The gNA variant of Embodiment 156, wherein the targeting sequence has 18 nucleotides

Embodiment 160. The gNA variant of Embodiment 156, wherein the targeting sequence has 17 nucleotides

Embodiment 161. The CasX variant of any one of Embodiments 1 to 40, wherein the CasX protein comprises a first domain from a first CasX protein and second domain from a second CasX protein different from the first CasX protein.

Embodiment 162. The CasX variant of Embodiment 161, wherein the first domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

Embodiment 163. The CasX variant of Embodiment 162, wherein the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

Embodiment 164. The CasX variant of any one of Embodiments 161 163, wherein the first and second domains are not the same domain.

Embodiment 165. The CasX variant of any one of Embodiments 161-164 wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 2.

Embodiment 166. The CasX variant of any one of Embodiments 161-164 wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 167. The CasX variant of any one of Embodiments 161-164, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 168. The CasX variant of any one of Embodiments 1 to 40 or 161-167, wherein the CasX protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second CasX protein different from the first CasX protein.

Embodiment 169. The CasX variant of Embodiment 168, wherein the at least one chimeric domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

Embodiment 170. The CasX variant of Embodiment 168 or 169, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 2.

Embodiment 171. The CasX variant of Embodiment 168 or 169, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 172. The CasX variant of Embodiment 168 or 169, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

Embodiment 173. The CasX variant of Embodiment 168, wherein the at least one chimeric domain comprises a chimeric RuvC domain.

Embodiment 174. The CasX variant of Embodiment 173, wherein the chimeric RuvC domain comprises amino acids 661 to 824 of SEQ ID NO: 1 and amino acids 922 to 978 of SEQ ID NO: 2.

Embodiment 175. The CasX variant of Embodiment 173, wherein the chimeric RuvC domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ ID NO: 1.

Embodiment 176. The gNA variant of any one of Embodiments 41-74, wherein the gNA comprises a first region from a first gNA and a second region from a second gNA.

Embodiment 177. The gNA variant of Embodiment 176, wherein the first region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

Embodiment 178. The gNA variant of Embodiment 176 or 177, wherein the second region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

Embodiment 179. The gNA variant of any one of Embodiments 176-178, wherein the first and second regions are not the same region.

Embodiment 180. The gNA variant of any one of Embodiments 176-179, wherein the first gNA comprises a sequence of SEQ ID NO: 4 and the second gNA comprises a sequence of SEQ ID NO: 5.

Embodiment 181. The gNA variant of any one of Embodiments 41-74 or 176-180, comprising at least one chimeric region comprising a first part from a first gNA and a second part from a second gNA.

Embodiment 182. The gNA variant of Embodiment 181, wherein the at least one chimeric region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

Embodiment 183. The gNA variant of Embodiment 182, wherein the first gNA comprises a sequence of SEQ ID NO: 4 and the second gNA comprises a sequence of SEQ ID NO: 5.

The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.

EXAMPLES Example 1: Assays Used to Measure sgRNA and CasX Protein Activity

Several assays were used to carry out initial screens of CasX protein and sgRNA DME libraries and engineered mutants, and to measure the activity of select protein and sgRNA variants relative to CasX reference sgRNAs and proteins.

E. coli CRISPRi Screen:

Briefly, biological triplicates of dead CasX DME Libraries on a chloramphenicol (CM) resistant plasmid with a GFP guide RNA on a carbenicillin (Carb) resistant plasmid were transformed (at >5× library size) into MG1655 with genetically integrated and constitutively expressed GFP and RFP (see FIG. 13A-13B). Cells were grown overnight in EZ-RDM+Carb. CM and Anhydrotetracycline (aTc) inducer. E. coli were FACS sorted based on gates for the top 1% of GFP but not RFP repression, collected, and resorted immediately to further enrich for highly functional CasX molecules. Double sorted libraries were then grown out and DNA was collected for deep sequencing on a highseq. This DNA was also re-transformed onto plates and individual clones were picked for further analysis.

E. coli Toxin Selection:

Briefly carbenicillin resistant plasmid containing an arabinose inducible toxin were transformed into E. coli cells and made electrocompetent. Biological triplicates of CasX DME Libraries with a toxin targeted guide RNA on a chloramphenicol resistant plasmid were transformed (at >5× library size) into said cells and grown in LB+CM and arabinose inducer. E. coli that cleaved the toxin plasmid survived in the induction media and were grown to mid log and plasmids with functional CasX cleavers were recovered. This selection was repeated as needed. Selected libraries were then grown out and DNA was collected for deep sequencing on a highseq. This DNA was also re-transformed onto plates and individual clones were picked for further analysis and testing.

Lentiviral Based Screen EGFP Screen:

Lentiviral particles were produced in HEK293 cells at a confluency of 70%-90% at time of transfection. Cells were transfected using polyethylenimine based transfection of plasmids containing a CasX DME library. Lentiviral vectors were co-transfected with the lentiviral packaging plasmid and the VSV-G envelope plasmids for particle production. Media was changed 12 hours post-transfection, and virus harvested at 36-48 hours post-transfection. Viral supernatants were filtered using 0.45 mm membrane filters, diluted in cell culture media if appropriate, and added to target cells HEK cells with an Integrated GFP reporter. Polybrene was supplemented to enhance transduction efficiency, if necessary. Transduced cells were selected for 24-48 hours post-transduction using puromycin and grown for 7-10 days. Cells were then sorted for GFP disruption & collected for highly functional sgRNA or protein variants (see FIG. 2). Libraries were then Amplified via PCR directly from the genome and collected for deep sequencing on a highseq. This DNA could also be re-cloned and re-transformed onto plates and individual clones were picked for further analysis.

Assaying Editing Efficiency of an HEK EGFP Reporter:

To assay the editing efficiency of CasX reference sgRNAs and proteins and variants thereof. EGFP HEK293T reporter cells were seeded into 96-well plates and transfected according to the manufacturer's protocol with lipofectamine 3000 (Life Technologies) and 100-200 ng plasmid DNA encoding a reference or variant CasX protein, P2A-puromycin fusion and the reference or variant sgRNA. The next day cells were selected with 1.5 μg/ml puromycin for 2 days and analyzed by fluorescence-activated cell sorting (FACS) 7 days after selection to allow for clearance of EGFP protein from the cells. EGFP disruption via editing was traced using an Attune NxT Flow Cytometer and high-throughput autosampler.

Example 2: Cleavage Efficiency of CasX Reference sgRNA

The reference CasX sgRNA of SEQ ID NO: 4 (below) is described in WO 2018/064371, the contents of which are incorporated herein by reference.

(SEQ ID NO: 4) 1 ACAUCUGGCG CGUUUAUUCC AUUACUUUGG AGCCAGUCCC AGCGACUAUG UCGUAUGGAC 61 GAAGCGCUUA UUIAUCGGAG AGAAACCGAU AAGUAAAACG CAUCAAAG.

It was found that alterations to the sgRNA reference sequence of SEQ ID NO: 4, producing SEQ ID NO: 5 (below) were able to improve CasX cleavage efficiency.

(SEQ ID NO: 5) 1 UACUGGCGCU UUUAUCUCAU UACUUUGAGA GCCAUCACCA GCGACUAUGU CGUAUGGGUA 61 AAGCGCUUAU UUAUCGGAGA GAAAUCCGAU AAAUAAGAAG CAUCAAAG.

To assay the editing efficiency of CasX reference sgRNAs and variants thereof, EGFP HEK293T reporter cells were seeded into 96-well plates and transfected according to the manufacturer's protocol with lipofectamine 3000 (Life Technologies) and 100-200 ng plasmid DNA encoding a reference CasX protein, P2A-puromycin fusion and the sgRNA. The next day cells were selected with 1.5 μg/ml puromycin for 2 days and analyzed by fluorescence-activated cell sorting (FACS) 7 days after selection to allow for clearance of EGFP protein from the cells. EGFP disruption via editing was traced using an Attune NxT Flow Cytometer and high-throughput autosampler.

When testing cleavage of an EGFP reporter by CasX reference and sgRNA variants, the following DNA encoding spacer target sequences were used:

E6 (TGTGGTCGGGGTAGCGGCTG; SEQ ID NO: 29) and E7 (TCAAGTCCGCCATGCCCGAA; SEQ ID NO: 30).

An example of the increased cleavage efficiency of the sgRNA of SEQ ID NO: 5 compared to the sgRNA of SEQ ID NO: 4 is shown in FIG. 5A. Editing efficiency of SEQ ID NO: 5 was improved 176% compared to SEQ ID NO: 4. Accordingly, SEQ ID NO: 5 was chosen as reference sgRNA for DME and additional sgRNA variant design, described below.

Example 3: Mutagenesis of CasX Reference gRNA Produces Variants with Improved Target Cleavage

DME of the sgRNA was achieved using two distinct PCR methods. The first method, which generates single nucleotide substitutions, makes use of degenerate oligonucleotides. These are synthesized with a custom nucleotide mix, such that each locus of the primer that is complementary to the sgRNA locus has a 97% chance of being the wild type base, and a 1% chance of being each of the other three nucleotides. During PCR, the degenerate oligos anneal to, and just beyond, the sgRNA scaffold within a small plasmid, amplifying the entire plasmid. The PCR product was purified, ligated, and transformed into E. coli. The second method was used to generate sgRNA scaffolds with single or double nucleotide insertions and deletions. A unique PCR reaction was set up for each base pair intended for mutation: In the case of the CasX scaffold of SEQ ID NO: 5, 109 PCRs were used. These PCR primers were designed and paired such that PCR products either were missing a base pair, or contained an additional inserted base pair. For inserted base pairs, PCR primers inserted a degenerate base such that all four possible nucleotides were represented in the final library.

Once constructed, both the protein and sgRNA DME libraries were assayed in a screen or selection as described in Example 1 to quantitatively identify mutations conferring enhanced functionality. Any assay, such as cell survival or fluorescence intensity, is sufficient so long as the assay maintains a link between genotype and phenotype. High throughput sequencing of these populations and validating individual variant phenotypes provided information about mutations that affect functionality as assayed by screening or selection. Statistical analysis of deep sequencing data provided detailed insight into the mutation landscape and mechanism of protein function or guide RNA function (see FIG. 3A-3B, FIG. 4A, FIG. 4B, FIG. 4C).

DME libraries sgRNA RNA variants were made using a reference gRNA of SEQ ID NO: 5, underwent selection or enrichment, and were sequenced to determine the fold enrichment of the sgRNA variants in the library. The libraries included every possible single mutation of every nucleotide, and double indels (insertion/deletions). The results are shown in FIGS. 3A-3B, FIG. 4A-4C, and Table 4 below.

To create a library of base pair substitutions using DME, two degenerate oligonucleotides that each bind to half of the sgRNA scaffold and together amplify the entire plasmid comprising the starting sgRNA scaffold were designed. These oligos were made from a custom nucleotide mix with a 3% mutation rate. These degenerate oligos were then used to PCR amplify the starting scaffold plasmid using standard manufacturing protocols. This PCR product was gel purified, again following standard protocols. The gel purified PCR product was then blunt end ligated and electroporated into an appropriate E. coli cloning strain. Transformants were grown overnight on standard media, and plasmid DNA was purified via miniprep.

To generate a library of small insertions and deletions, PCR primers were designed such that the PCR products resulting from amplification of the plasmid comprising the base sgRNA scaffold would either be missing a base pair, or contain an additional inserted base pair. For inserted base pairs, PCR primers were designed in which a degenerate base has been inserted, such that all four possible nucleotides were represented in the final library of pooled PCR products. The starting sgRNA scaffold was then PCR amplified with each set of oligos as their own reaction. Each PCR reaction contained five possible primers, although all primers annealed to the same sequence. For example, Primer 1 omitted a base, in order to create a deletion. Primers 2, 3, 4, and 5 inserted either an A, T, G, or C. However, these five primers all annealed to the same region and hence could be pooled in a single PCR However, PCRs for different positions along the sgRNA needed to be kept in separate tubes, and 109 distinct PCR reactions were used to generate the sgRNA DME library.

The resulting 109 PCR products were then run on an agarose gel and excised before being combined and purified. The pooled PCR products were blunt ligated and electroporated into E. coli. Transformants were grown overnight on standard media with an appropriate selectable marker, and plasmid DNA was purified via miniprep. Having created a library of all single small indels, the steps of PCR amplifying the starting plasmid with each set of oligos, purifying, blunt end ligating, transforming into E col and mini-prepping can be repeated to obtain a library containing most double small indels. Combining the single indel library and double indel library at a ratio of 1:1000 resulted in a library that represented both single and double indels.

The resulting libraries were then combined and passed through the DME screening and/or selection process to identify variants with enhanced cleavage activity. DME libraries were screened using toxin cleavage and CRISPRi repression in E. coli, as well as EGFP cutting in lentiviral-transfected HEK293 cells, as described in Example 1. The fold enrichment of scaffold variants in DME libraries that have undergoing screening/selection followed by sequencing is shown below in Table 4. The read counts associated with each of the below sequences in Table 4 were determined (‘annotations’, ‘seq’). Only sequences with at least 10 reads across any sample were analyzed to filter from 15 Million to 600 K sequences. The below ‘seq’ gives the sequence of the entire insert between the two 5′ random 5mer and the 3′ random 5mer. ‘seq_short’ gives the anticipated sequence of the scaffold only. The mutations associated with each sequence were determined through alignment (‘muts’). All modifications are indicated by their [position (0-indexed)].[reference base].[alternate base]. Position 0 indicates the first T of the transcribed gRNA. Sequences with multiple mutations are semicolon separated. The column muts_1indexed, gives the same information but 1-indexed instead of 0-indexed. Each of the modifications are annotated (‘annotated_variants’), as being a single substitution/insertion/deletion, double substitution/insertion/deletion, single_del_single_sub (a deletion and an adjacent substitution), a single_sub_single_ins (a substitution and adjacent insertion), ‘outside_ref’ (indicates that the modification is outside the transcribed gRNA), or ‘other’ (any larger substitution/insertion/deletion or some combination thereof). An insertion at position i indicates an inserted base between position i−1 and i (i.e. before the indicated position). To note about variant annotation: a deletion of any one of a consecutive set of bases can be attributed to any of those bases. Thus, a deletion of the T at position −1 is the same sequence as a deletion of the T at position 0. ‘counts’ indicates the sequencing-depth normalized read count per sequence per sample. Technical replicates were combined by taking the geometric mean. ‘log 2enrichment’ gives the median enrichment (using a pseudocount of 10) across each context, or across all samples, after merging for technical replicates. The naive read count was averaged (geometric) between the D2_N and D3_N samples. Finally, the ‘log 2enrichment_err’ gives the ‘confidence interval’ on the mean log 2 enrichment. It is the standard deviation of the enrichment across samples*2/sqrt of the number of samples. Below, only the sequences with median log 2enrichment−log 2enrichment_err>0 are shown (2704/614564 sequences examined).

In Table 4, CI indicates confidence interval and MI indicates median enrichment, which indicates enhanced activity.

TABLE 4 Median Enrichment of DME Scaffold Variants SEQ 95% index ID NO muts_lindexed MI CI 7240543 412 27.-.C;76.G.- 3.390 2.040 7240150 413 27.-.C;75.-.C 3.111 1.862 2584994 414 0.T.-;2.A.C;27.-.C 2.997 1.806 2618163 415 0.T.-;2.A.C;55.-.G 2.915 0.725 2655870 416 2.A.C;0.T.-;76.GG.-A 2.903 0.391 2762330 417 2.A.C;0.T.-;55.-.T 2.857 1.290 7247368 418 27.-.C;86.C.- 2.835 1.637 2731505 419 2.A.C;0.T.-;75.-.G 2.795 0.625 2729600 420 2.A.C;0.T.-;76.-.T 2.791 0.628 2701142 421 2.A.C;0.T.-;87.-.T 2.768 0.559 2659588 422 2.A.C;0.T.-;75.-.C 2.733 0.477 2582823 423 0.T.-;2.A.C;27.-.A 2.729 1.669 3000598 424 1.TA.--;76.G.- 2.704 0.439 10565036 425 15.-.T;74.-.T 2.681 0.808 9696472 426 28.-.T;76.GG.-T 2.681 1.715 2674674 427 2.AC;0.T.-;86.-.C 2.650 0.772 7254130 428 27.-.C;75.CG.-T 2.629 1.755 2977442 429 1.TA.--;55.-.G 2.629 0.887 2661951 430 2.A.C;0.T.-;76.G.- 2.627 0.432 1937646 431 2.A.C;0.TT.--;75.-.C 2.626 1.328 2232796 432 0.T.-;55.-.G 2.607 0.777 2714418 433 0.T.-;2.C;81.GA.-T 2.595 0.443 2700142 434 2.A.C.0.T-;87.-.G 2.582 0.608 2667512 435 2.A.C;0.T.-;77.GA.-- 2.577 0.588 7239606 436 27.-.C;76.-.A 2.566 1.441 10563356 437 15.-.T;75.-.G 2.557 1.056 7181049 438 27.-.A;75.-.C 2.543 1.893 2720034 439 2.A.C;0.T.-;78.- 2.531 0.492 2265581 440 0.T.-;86.-.C 2.520 0.504 2256355 441 0.T.-;76.GG.-C 2.516 0.942 7251229 442 27.-.C;76.-.G 2.516 1.793 10281529 443 17.-.T;76.GG.-A 2.515 1.104 2299702 444 0.T.-;74.-.T 2.504 0.392 2670445 445 2.A.C;0.T.-;85.T.- 2.499 1.225 2258816 446 0.T.-;76.G.- 2.494 0.475 7241311 447 27.-.C;77.GA.-- 2.493 1.595 2658150 448 2.A.C;0.T.-;76.GG.-C 2.492 0.585 2734378 449 2.A.C;0.T.-;74.-.T 2.490 0.485 2723181 450 2.A.C;0.T.-;76.-.G 2.488 0.421 2288202 451 0.T.-;81.GA.-T 2.487 0.591 2278172 452 0.T.-;89.-.C 2.486 0.690 2997382 453 1.TA.--;76.GG.-A 2.465 1.066 2255017 454 0.T.-;76.GG.-A 2.463 0.422 2257399 455 0.T.-;75.-.C 2.460 0.676 12183183 456 2.A.-;81.GA.-T 2.459 0.736 7252067 457 27.-.C;76.GG.-T 2.459 2.062 10525083 458 15.-.T;75.-.C 2.448 1.006 7253869 459 27.-.C;74.-.T 2.439 1.638 4303777 460 4.T.-;76..T 2.435 0.782 2741395 461 2.A.C;0.T.-;73.A.- 2.435 0.633 7250940 462 27.-.C;78.A.- 2.423 2.064 4302595 463 4.T.-;76.GG.-T 2.422 0.850 4275786 464 4.T.-;87.-.T 2.420 1.019 2650980 465 2.A.C;0.T.-;74.-.C 2.414 0.462 2458336 466 1.TA.--;3.C.A;76.G.- 2.411 1.089 10284144 467 17.-.T;76.G.- 2.406 1.638 2726809 468 2.A.C;0.T.-;76.G.-;78.A.T 2.400 0.556 2280896 469 0.T.-;87.-.T 2.398 0.560 2673790 470 2.A.C;0.T.-;88.G.- 2.398 1.017 3188700 471 0.T.-;2.A.G;27.-.C 2.394 1.732 9632434 472 16.------------ 2.394 1.141 .CTCATTACTTTG;75.-.G 3029757 473 1.TA.--;78.A.- 2.392 0.524 2728393 474 2.A.C.0.T.-;76.GG.-T 2.390 0.714 2300381 475 0.T.-;75.CG.-T 2.385 0.948 2279969 476 0.T.-;86.C.- 2.382 0.404 2260011 477 0.T.-;77.-.C 2.379 0.608 2248579 478 0.T.-;72-.C 2.377 0.743 12075394 479 2.A.-;55.-.G 2.377 0.679 9602743 480 28.-.C;76.GG.-C 2.376 1.681 2736722 481 2.A.C;0.T.-;73.AT.-C 2.374 1.104 12117240 482 2.A.-;76.GG-A 2.372 0.429 10307397 483 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558 4.T.-;89.-.A 2.117 0.998 2688981 559 2.A.C;0.T.-;99.-.G 2.112 0.980 2995452 560 1.TA.--;74.-.G 2.112 0.611 12114782 561 2.A.-;75.-.A 2.110 0.500 2993173 562 1.TA.--;73.-.A 2.104 0.697 1978344 563 0.T.C;87.-.G 2.100 0.870 4294004 564 4.T.-;78.-.C 2.099 0.595 10568306 565 15.-.T;73.A.- 2.096 0.741 10561545 566 15.-.T;76.GG.-T 2.095 0.554 2713433 567 2.A.C;0.T.-;82.AA.-T 2.094 0.560 1863579 568 0.TT.--;75.-.G 2.086 0.787 3006303 569 1.TA.-;88.G.- 2.086 0.537 4236935 570 4.T.-;76.G.- 2.081 0.919 12138801 571 2.A.-;89.-.A 2.080 1.115 12164760 572 2.A.-;89.-.T 2.080 0.316 10288787 573 17.-.T;86.-.C 2.080 0.927 2664128 574 0.T.-;2.A.C;77.-.C 2.079 0.379 2663861 575 0.T.-;2.A.C;76.G.-;78.A.C 2.078 0.700 2726063 576 0.T.-;2.A.C;78.A.T 2.078 0.972 4232837 577 4.T.-;76.GG-C 2.069 0.580 3001194 578 1.TA.--;77.-.A 2.063 0.629 2048069 579 0.TT.--;2.A.G;76.G.- 2.059 1.413 2653681 580 2.A.C;0.T.-;75.-.A 2.052 0.427 2265126 581 0.T.-;88.G.- 2.050 0.557 2739399 582 0.T.-;2.A.C;73.A.G 2.049 1.003 7250543 583 27.-.C;78.-.C 2.047 1.480 2747651 584 0.T.-;2.A.C;66.CT.-- 2.047 0.900 12437734 585 1.TAC.---;78.A.- 2.043 0.615 2826230 586 0.T.-;2.A.C;15.-.T 2.042 0.538 2709008 587 2.A.C.;0.T.-;82.A.-;84.A.T 2.037 1.246 3005336 588 1.TA.--;86.-.A 2.034 0.483 4301274 589 4.T.-;76.G.-;78.A.T. 2.028 0.873 3018865 590 1.TA.--;86.C.- 2.025 0.616 2699310 591 2.A.C;0.T.-;86.C- 2.023 0.564 2279026 592 0.T.-;89.A.- 2.022 1.568 7248209 593 27.-.C;82.A.- 2.022 1.627 10562113 594 15.-.T;76.-.T 2.020 0.858 7181373 595 27.-.A;76.G.- 2.014 1.908 10559019 596 15.-.T;76.-.G 2.014 0.753 3018452 597 1.TA.--;88.-.T 2.013 0.626 12118457 598 2.A.-;76.-.A 2.011 1.170 2805043 599 2.A.C;0.T.-;28.-C 2.010 1.524 4242379 600 4.T.-;77.GA.-- 2.008 0.985 2259846 601 0.T.-;76.G.-;78.A.C 2.005 0.640 6462092 602 16.-.C;87.-.A 2.001 0.983 4312495 603 4.T.-;73.AT.-G 1.997 0.708 2668714 604 0.T.-;2.A.C;81.GA.-C 1.996 0.678 2294477 605 0.T.-;78.AG.-T 1.994 0.703 12198135 606 2.A.-;77.-.T 1.994 1.433 4238150 607 4.T.-;77.-.A 1.993 0.762 3019738 608 1.TA.--.87.-.T 1.992 0.532 2352050 609 0.T.-;17.-.T 1.991 0.852 2705912 610 2.A.C;0.T.-;83.-.C 1.990 0.585 6478822 611 16.-.C;74.-.T 1.989 0.477 2665913 612 2.A.C;0.T.-;79.GA.-C 1.987 1.186 3331447 613 2.A.G;0.T.-;76.GG.-T 1.985 0.958 3186538 614 2.A.G;0.T.-;27.-.A 1.983 1.530 2738784 615 2.A.C.0.T.-;73.AT.-G 1.977 0.623 7832272 616 55.-.G 1.977 0.882 4297458 617 4.T.-;76.-.G 1.976 0.997 3334291 618 2.A.G;0.T.-;75.-.G 1.975 0.654 2212416 619 0.T.-;27.-.C 1.974 1.458 8752897 620 55.-.T;76.G.- 1.972 0.468 2293333 621 0.T.-;76.-.G 1.970 0.514 7180386 622 27.-.A;76.GG.-A 1.969 1.667 2996180 623 1.TA.--;75.-.A 1.967 0.476 7238423 624 27.-.C;74.T.- 1.963 1.563 2261752 625 0.T.-;77.GA.-- 1.962 0.503 10282247 626 17.-.T;76.GG.-C 1.960 0.719 4230973 627 4.T.-;76.GG.-A 1.958 0.723 4276520 628 4.T.-;86.-.G 1.958 0.901 2675193 629 0.T.-;2.A.C;88.GA.-C 1.957 0.878 13101476 630 -1.GT.--;75.-.G 1.952 0.439 7203209 631 27.G.-;76.GG.-C 1.952 1.709 2724398 632 0.T.-;2.A.C;78.A.G 1.947 0.801 10309365 633 17.-.T;78.-.T 1.947 1.542 10520418 634 15.-.T;74.T.- 1.945 0.728 10300394 635 17.-.T;87.-.G 1.944 1.037 4248302 636 4.T.-;88.G.- 1.937 0.857 7240856 637 27.-.C;76.G.-;78.A.C 1.937 1.188 4313003 638 4.T.-;73.A.G 1.935 0.688 2467599 639 1.TA.--;3.C.A;76.GG.-T 1.923 1.105 2279202 640 0.T.-;89.-.T 1.921 0.709 2259410 641 0.T.-;77.-.A 1.920 0.417 4305674 642 4.T.-;75.-.G 1.915 1.089 6459602 643 16.-.C;7G- 1.915 0.642 2701869 644 0.T.-;2.A;86.-.G 1.914 0.477 2252978 645 0.T.-;74.-.G 1.911 0.602 6470049 646 16.-.C;87.-.G 1.910 0.715 12134362 647 2.A.-;86.-.A 1.907 0.661 12209524 648 2.A.-;73.A.C 1.901 1.154 2260529 649 0.T.-;79.G.- 1.900 0.829 2690549 650 0.T.-;2.A.C;98.-.T 1.899 0.954 10073100 651 19.-.T;88.G.- 1.898 0.782 4239969 652 4.T.-;79.G.- 1.898 0.794 3026047 653 1.TA.--;81.GA.-T 1.896 0.555 3003294 654 1.TA.--;77.GA.-- 1.896 0.506 12121216 655 2.A.-;75.-.C 1.895 0.610 2696635 656 0.T.-;2.A.C;89.AT.-G 1.894 0.882 12130978 657 2.A.-;81.GA.-C 1.891 0.936 6475473 658 16.-.C;78.A.- 1.889 0.581 1853356 659 0.TT.--;76.G.- 1.885 0.802 8544082 660 75.-.G;87.-.G 1.884 0.536 2884429 661 1.-.C;76.G.- 1.884 0.673 63955 662 17.-.A;76.-.G 1.882 0.843 2746170 663 2.A.C;0.T.-;66.CT.-G 1.880 0.517 4226314 664 4.T.-;74.-.C 1.874 0.901 6304607 665 16.-.A;76.G.- 1.873 0.523 2583788 666 0.T.-;2.A.C;27.G.- 1.873 1.388 2255694 667 0.T.-;76.-.A 1.869 0.837 7249882 668 27.-.C;80.A.- 1.867 1.645 10069481 669 19.-.T;75.-.C 1.864 0.645 2643173 670 0.T.-;2.A.C;70.T.- 1.864 1.689 12749699 671 0.-.T;75.-.G 1.863 0.757 7208859 672 27.G.-87.-.G 1.862 1.687 4271233 673 4.T.-;89.-.C 1.854 0.839 6455215 674 16.-.C;73.-.A 1.850 0.825 2816525 675 0.T.-;2.A.C;19.-.T 1.848 0.369 2292594 676 0.T.-;78.A.- 1.846 0.313 2287708 677 0.T.-;82.AA.-T 1.846 0.408 2721779 678 2.A.C;0.T.-;78.A.- 1.842 0.677 1945942 679 0.TT.--;2.A.C;75.-.G 1.842 1.271 12111705 680 2.A.-;74.-.C 1.841 0.669 2567750 681 0.T.-;2.A.C;16.-.C 1.840 0.427 2463364 682 1.TA.--;3.C.A;87.-.G 1.839 0.821 3031594 683 1.TA.--;78.AG.-T 1.839 0.620 10199376 684 18.-.G;75.-.G 1.837 1.238 4272444 685 4.T.-;89.A.- 1.837 0.998 9610551 686 28.-.C;78.A.- 1.836 1.802 2737747 687 0.T.-;2.A.C;73.A.C 1.833 1.293 12113430 688 2.A.-;74.-.G 1.828 0.753 10530413 689 15.-.T;85.TC.-G 1.825 1.155 12176759 690 2.A.-;83.-.T 1.824 1.046 12127185 691 2.A.-;79.G.- 1.824 0.606 4288099 692 4.T.-;81.GA.-T 1.824 0.753 12196850 693 2.A.-;78.A.T 1.821 1.086 6457366 694 16.-.C;75.-.A 1.821 0.638 12105140 695 2.A.-;72.-.C 1.818 0.700 1944577 696 0.TT.--;2.A.C;78.A.- 1.817 1.170 4293546 697 4.T.-;78.AG.-C 1.816 1.015 9996838 698 19.-.G;74.-.T 1.814 0.800 10301024 699 17.-.T;86.-.G 1.814 0.967 2308228 700 0.T.-;66.C.- 1.811 0.756 7835938 701 55.-.G;75.-.G 1.811 1.112 3005841 702 1.TA.--;87.-.A 1.811 0.806 12169698 703 2.A.-;86.-.G 1.808 0.857 3028597 704 1.TA.--;78.AG.-C 1.803 0.743 7191855 705 27.-.A;75.CG.-T 1.802 1.430 9972503 706 19.-.G;74.T.- 1.802 0.750 4026979 707 3.-.C;75.-.G 1.802 1.374 7180118 708 27.-.A;75.-.A 1.801 1.525 10081203 709 19.-.T;86.C.- 1.799 0.502 10532156 710 15.-.T;86.-.C 1.797 1.070 2749667 711 2.A.C;0.T.-;65.GC.-T 1.795 0.642 129228 712 2.A.-;90.-.C 1.794 1.201 10288547 713 17.-.T.88.G.- 1.794 1.193 4331367 714 4.T.-;55.-.T 1.793 0.481 2725463 715 2.A.C;0.T.-;78.-.T 1.792 0.507 2718857 716 0.T.-;2.A.C;79.GA.-T 1.792 0.900 2247247 717 0.T.-;72.-.A 1.792 0.887 12125011 718 2.A.-;77.-.A 1.786 0.527 4225246 719 4.T.-;74.T.- 1.786 0.629 12165722 720 2.A.-;88.-.T 1.786 1.273 2733129 721 0.T.-;2.A.C;75.C.- 1.786 0.561 2469676 722 1.TA.--;3.C.A.73.A.- 1.785 1.174 3018172 723 1.TA.--;89.-.T 1.785 0.757 12196049 724 2.A.-;78.-.T 1.782 0.754 9612063 725 28.-.C;74.-.T 1.782 1.618 10547909 726 15.-.T;86.-.G 1.781 0.818 12194342 727 2.A.-;78.A.-;80.A.- 1.780 1.289 4228855 728 4.T.-;75.-.A 1.776 0.897 10546613 729 15.-.T;86.C.- 1.776 0.859 10547538 730 15.-.T;87.-.T 1.772 1.080 10519772 731 15.-.T;73.-.A 1.771 0.624 8510297 732 77.G.T 1.770 1.239 12119606 733 2.A.-;76.GG.-C 1.768 1.110 2669299 734 0.T.-;2.A.C.85.TC.-A 1.767 0.842 6469807 735 16.-.C;86.C.- 1.765 0.759 10197299 736 18.-.G;76.-.G 1.764 0.832 3344225 737 2.A.G;0.T.-;73.A.- 1.762 1.216 2456917 738 1.TA.--;3.C.A;75.-.A 1.761 1.203 10307233 739 17.-.T;78.AG.-C 1.760 1.101 12314352 740 2.A.-;15.-.T 1.758 0.436 12177388 741 2.A.-;82.AA.-- 1.751 0.615 2694455 742 0.T.-;2.A.C;91.A.-;93.A.G 1.751 1.015 3040066 743 1.TA.--;73.A.- 1.750 0.690 10081633 744 19.-.T;87.-.T 1.750 0.917 4246508 745 4.T.-;86.-.A 1.749 0.939 4301580 746 4.T.-;77.-.T 1.744 0.701 10181172 747 18.-.G;75.-.A 1.743 1.016 12200668 748 2.A.-;76.-.T 1.741 0.873 10524336 749 15.-.T;76.GG.-C 1.738 0.390 3007212 750 1.TA.--;89.-.A 1.738 1.072 10526271 751 15.-.T;76.G.- 1.738 1.098 10561166 752 15.-.T;77.-.T 1.737 0.745 2663037 753 2.A.C;0.T.-;77.-.A 1.732 0.417 12136525 754 2.A.;88.G.- 1.731 0.578 8758832 755 55.-.T;78.A.- 1.731 0.641 1864295 756 0.TT.--;75.CG.-T 1.729 0.424 10550736 757 15.-.T;82.A.-;84.A.G 1.728 0.888 2657071 758 2.A.C;0.T.-;76.-.A 1.728 1.206 2059338 759 0.TT.--;2.A.G;75.-.G 1.725 1.054 12182224 760 2.A.-;82.AA.-T 1.722 0.599 2671130 761 2.A.C;0.T.-;85.TC.-G 1.721 0.884 4200182 762 4.T.-;55.-.G 1.721 1.233 2281298 763 0.T.-;86.-.G 1.720 0.460 7182097 764 27.-.A;77.GA.-- 1.719 1.318 2251662 765 0.T.-;74.T.- 1.719 0.428 1904870 766 0.TTA.---;3.C.A;76.G.- 1.715 1.345 10553996 767 15.-.T;81.GA.-T 1.715 0.963 10202590 768 18.-.G;73.A.- 1.715 0.822 3028839 769 1.TA.--;78.-.C 1.713 0.450 3304552 770 0.T.-;2.A.G;89.-.T 1.713 0.767 4247308 771 4.T.-;87.-.A 1.711 0.766 4318521 772 4.T.-;66.CT.-G 1.710 0.957 7247759 773 27.-.C;86.-.G 1.710 1.198 10198320 774 18.-.G;76.GG.-T 1.709 0.701 2457655 775 1.TA.-.3.C.A;76.GG.-C 1.709 1.260 3032520 776 1.TA.--;76.G.-;78.A.T 1.709 0.754 2702792 777 0.T.-2;A.C;86.CC.-T 1.709 0.742 12171374 778 2.A.-;84.AT.-- 1.709 1.239 10192666 779 18.-.G;87.-.G 1.706 0.672 2642318 780 2.A.C;0.T.-;72.-.A 1.703 0.651 2718074 781 2.A.C;0.T.-;77.GA.--82.A.T 1.700 1.191 12191670 782 2.A.-;78.A.- 1.697 0.819 2456219 783 1.TA--;3.C.A;74.T.- 1.696 1.260 2457365 784 1.TA.--;3.C.A;76.GG.-A 1.695 0.951 8538180 785 75.-.G 1.695 0.416 3020581 786 1.TA.--;86.CC.-T 1.693 1.160 10281916 787 17.-.T;76.-.A 1.693 0.649 2707684 788 0.T.-;2.A.C;82.A.-;84.A.G 1.692 1.346 2676761 789 0.T.-;2.A.C;90.-.G 1.689 1.000 7213979 790 27.G.-;75.CG.-T 1.689 1.195 2459101 791 1.TA.--;3.C.A;77.GA.-- 1.687 0.967 8123571 792 75.-.C.86.-.C 1.686 0.454 12207287 793 2.A.-;75.CG.-T 1.685 0.564 2740245 794 2.A.C;0.T.-;70.-.T 1.685 1.013 10531744 795 15.-.T;88.G.- 1.685 1.172 2669798 796 2.A.C;0.T.-;82.-A 1.684 0.486 2294771 797 0.T.-;78.-.T 1.684 0.366 7213033 798 27.G.-;76.GG.-T 1.687 1.554 7829581 799 55.-.G;76.G.- 1.682 1.158 2808092 800 0.T.-;2.A.C;28.-.T 1.680 1.571 2960043 801 1.TA.--;27.-.C 1.676 1.353 10506564 802 15.-.T;55.-.G 1.675 1.443 4315349 803 4.T.-;73.A.T 1.668 0.705 2705067 804 2.A.C;0.T.-;82.A.- 1.668 0.498 3330280 805 0.T.-;2.A.G;76.G.-;78.A.T 1.667 0.948 9630969 806 16.------------ 1.665 1.315 .CTCATTACTTTG;75.-.A 12173513 807 2.A.-;82.A.- 1.664 0.734 3280346 808 0.T.-;2.A.G;87.-.A 1.663 1.204 7238549 809 27.-.C;74.-.C 1.661 1.215 8154695 810 76.G.-;78.A.C 1.661 0.368 10516784 811 15.-.T;72.-.A 1.660 0.597 10307953 812 17.-.T;78.A.- 1.660 0.824 12432835 813 1.TAC.---;75.-.C 1.654 0.814 12193344 814 2.A.-;76.-.G 1.654 0.664 2297191 815 0.T.-;76.-.T 1.652 0.458 2126158 816 0.TTA.---;3.C.G;87.-.G 1.650 1.318 2283617 817 0.T.-;83.-.C 1.649 1.421 2654520 818 2.A.C;0.T.-;75.CG.-A 1.647 0.574 3332543 819 0.T.-;2.A.G;76.-.T 1.645 0.844 9604425 820 28.-.C;88.G.- 1.644 1.218 12109255 821 2.A.-;73.-.A 1.644 0.930 12438229 822 1.TAC.---;76.GG.-T 1.642 0.689 8153054 823 77.G.C 1.641 1.385 10308482 824 17.-.T;76.-.G 1.641 1.127 10300026 825 17.-.T;86.C.- 1.641 1.228 2715234 826 2.A.C;0.T.-;80.AG.-C 1.640 1.476 10532541 827 15.-.T;90.T.- 1.640 1.020 12721860 828 0.-.T;76.G.- 1.640 0.367 2460008 829 1.TA.--;3.C.A;86.-.C 1.639 0.936 2264044 830 0.T.-;86.-.A 1.639 0.512 12188811 831 2.A.-;78.AG.-C 1.638 0.776 12432569 832 1.TAC.---;76.GG.-A 1.637 0.883 9602947 833 28.-.C;75.-.C 1.636 1.558 2994003 834 1.TA.--;74.T.- 1.634 0.542 12213405 835 2.A.-;73.A.- 1.634 0.736 2719575 836 0.T.-;2.A.C;78.AG.-C 1.633 0.446 2123173 837 0.TTA.---;3.C.G;76.G.- 1.632 1.511 10086342 838 19.-.T;78.-.C 1.631 0.477 12236371 839 2.A.-;55.-.T 1.630 0.850 6473588 840 16.-.C;81.GA.-T 1.628 0.398 7240999 841 27.-.C;79.G.- 1.628 1.310 12189370 842 2.A.-;78.-.C 1.625 0.715 3005003 843 1.T.A.--;85.TC.-G 1.625 0.820 10185851 844 18.-.G;86.-.C 1.622 0.770 2725020 845 0.T.-;2.A.C;78.AG.-T 1.622 0.696 12212274 846 2.A.-;70.-.T 1.621 1.038 8470264 847 78.-.C 1.617 0.272 2286841 848 0.T.-;82.AA.-G 1.617 0.606 7241506 849 27.-.C;81.GA.-C 1.617 1.112 12163987 850 2.A.-;89.A.G 1.617 0.718 3364655 851 0.T.-;2.A.G;55.-.T 1.615 1.131 1904677 852 0.TTA.---;3.C.A;75.-.C 1.614 0.965 2712438 853 2.A.C;0.T.-;82.-.T 1.612 0.769 14645004 854 -29.A.C;0.T.-;2.A.C;76.G.- 1.610 0.433 10322550 855 17.-.T;55.-.T 1.608 0.835 10304965 856 17.-.T;82.AA.-T 1.606 1.006 10279228 857 17.-.T;74.-.C 1.603 0.965 3263089 858 2.A.G;0.T.-;74.-.G 1.603 0.944 2282393 859 0.T.-;82.A.-;85.T.G 1.602 1.047 2463251 860 1.TA.--;3.C.A;86.C.- 1.598 0.959 2459897 861 1.TA.--;3.C.A;88.G.- 1.596 0.725 1852430 862 0.TT.--;76.GG.-A 1.596 0.848 10305251 863 17.-.T;81.GA.-T 1.593 1.079 9603994 864 28.-.C;85.TC.-A 1.593 1.339 4319798 865 4.T.-;66.CT.-- 1.593 0.719 3042484 866 1.TA.--;66.CT.-G 1.592 0.578 8544184 867 75.-.G;87.-.T 1.592 0.631 2709867 868 2.A.C;0.T.-;82.AA.-C 1.500 0.506 3439310 869 0.T.-;2.A.G;15.-.T 1.589 0.341 2718364 870 0.T.-;2.A.C;80.A.T 1.588 1.149 4223967 871 4.T.-;73.-.A 1.587 0.646 4271617 872 4.T.-;89.AT.-G 1.587 1.233 10460510 873 16.C.-;76.GG.-A 1.587 0.788 4227764 874 4.T.-;74.-.G 1.586 0.680 9994855 875 19.-.G;76.GG.-T 1.585 0.779 3272821 876 2.A.G;0.T.-;76.G.-;78.A.C 1.583 0.912 12110798 877 2.A.-;74.T.- 1.582 0.659 1975319 878 0.T.C;76.G.- 1.581 0.610 10316332 879 17.-.T;73.A.- 1.581 0.902 2720616 880 0.T.-;2.A.C;78.A.C 1.581 0.565 8753785 881 55.-.T;86.-.C 1.581 0.908 8112378 882 76.-.A 1.580 0.965 2819005 883 0.T.-;2.A.C;18.-.G 1.579 0.491 8357828 884 87.-.G 1.579 0.261 6477023 885 16.-.C;76.GG.-T 1.577 0.802 12737747 886 0.-.T;87.-.G 1.577 0.587 12309294 887 2.A.-;17.-.T 1.576 0.644 2252133 888 0.T.-;74.-.C 1.576 0.340 10567192 889 15.-.T;73.AT-G 1.575 0.657 3261438 890 2.A.G;0.T.-;74.-.C 1.575 0.783 15169229 891 -29.A.G;75.-.G 1.574 0.382 6128804 892 14.-.A;76.GG.-T 1.574 0.980 12197720 893 2.A.-;76.G.-;78.A.T. 1.573 0.893 3326919 894 2.A.G;0.T.-;76.-.G 1.573 0.783 12164376 895 2.A.-;89.A.- 1.572 1.400 2990209 896 1.TA.--;70.T.- 1.571 1.474 8538220 897 75.-.G;132.G.T 1.571 0.465 10068467 898 19.-.T;76.GG.-A 1.570 0.904 9697533 899 28.-.T;75.CG.-T 1.569 1.330 2958993 900 1.TA.--;27.-.A 1.568 1.255 3001629 901 1.TA.--;76.G.-;78.A.C 1.566 0.524 4291732 902 4.T.-;77.GA.--;82.A.T 1.565 1.310 4238868 903 4.T.-;76.G.-;78.A.C 1.564 0.830 3306461 904 0.T.-2.A.G;87.-.G 1.564 0.717 1937976 905 2.A.C;0.TT.--;76.G.- 1.560 1.463 4172716 906 4.T.-;27.-.C 1.558 1.388 12185288 907 2.A.-;80.A.- 1..557 0.706 14813579 908 -29.A.C;75.-.G 1.557 0.415 2468675 909 1.TA.--;3.C.A;75.CG-T 1.553 0.931 12195510 910 2.A.-78.AG.-T 1.550 0.887 4285997 911 4.T.-;82.AA.-G 1.549 0.782 3275841 912 2.A.G;0.T.-;77.GA.-- 1.549 0.526 3018032 913 1.TA.--89.A.- 1.549 1.114 2301817 914 0.T.-;73.A.C 1.549 0.917 3305057 915 0.T.-;2.A.G;88.-.T 1.548 0.420 2122618 916 0.TTA.---;3.C.G;76.GG.-A 1.548 1.094 2289325 917 0.T.-;80.A.- 1.547 0.393 4291562 918 4.T.-;80.AG.-T 1.547 1.017 10557226 919 15.-.T;78.-.C 1.545 0.975 12748115 920 0.-.T;76.GG.-T 1.545 0.710 3026518 921 1.TA.--;80.AG.-C 1.544 1.241 10545028 922 15.-.T;89.-.C 1.542 0.579 3416823 923 0.T.-;2.A.G;28.-.C 1.539 1.436 9976094 924 19.-.G;76.G.- 1.539 0.749 1852751 925 0.TT.--;76.GG.-C 1.537 0.770 4314686 926 4.T.-;73.A.- 1.536 1.014 6470272 927 16.-.C;87.-.T 1.536 0.597 2673006 928 0.T.-;2.A.C;87.C.A 1.535 0.804 12137377 929 2.A.-;86.-.C 1.535 0.546 12184036 930 2.A.-;80.AG.-C 1.532 1.352 10285242 931 17.-.T;77.-.C 1.530 1.164 2263017 932 0.T.-;82.-.A 1.530 0.468 12163286 933 2.A.-;89.AT.-G 1.529 1.001 2706481 934 2.A.C;0.T.-;82.A.-;84.A.C 1.528 1.209 4320578 935 4.T.-66.C.- 1.527 0.995 3004121 936 1.TA.--;85.TC.-A 1.526 0.698 3269260 937 2.A.G;0.T.-;75.-.C 1.522 0.739 7835518 938 55.-.G;76.-.G 1.519 0.935 10195401 939 18.-.G;81.GA.-T 1.519 0.776 6477333 940 16.-.C;76.-.T 1.516 0.627 4171307 941 4.T.-;27.-.A 1.514 1.234 10299590 947 17.-.T;88.-.T 1.513 1.296 6478447 943 16.-.C;75.C.- 1.512 0.508 4249490 944 4.T.-.;88.GA.-C 1.512 0.737 12220656 945 2.A.-;66.C.- 1.512 1.055 7240739 946 27.-.C;77.-.A 1.512 1.178 10315246 947 17.-.T;73.AT.-G 1.511 1.010 1944754 948 0.TT.--;2.A.C;76.-.G 1.511 1.156 3337255 949 2.A.G;0.T.-;74.-.T 1.510 0.678 6362999 950 17.-.A.76.G.- 1.509 1.043 3017407 951 1.TA.--;89.-.C 1.509 0.465 9973601 952 19.-.G;75.-.A 1.503 0.894 12186826 953 2.A.-;80.AG.-T 1.501 0.813 3035711 954 1.TA.--;75.C.- 1.500 0.592 8526584 955 76.-.T 1.499 0.320 2211100 956 0.T.-;27.-.A 1.499 1.300 8558515 957 74.-.T 1.499 0.244 4321895 958 4.T.-;65.GC.-T 1.498 0.661 12204638 959 2.A.-;75.C.- 1.496 0.655 8118238 960 76.GG.-C 1.495 0.555 2348592 961 0.T.-;19.-.T 1.493 0.463 3282394 962 0.T.-;2.A.G;88.GA.-C 1.491 1.144 9974216 963 19.-.G;76.GG.-A 1.490 0.650 3435006 964 0.T.-;2.A.G;17.-.T 1.488 0.572 2291281 965 0.T.-;78.AG.-C 1.486 0.722 3013663 966 1.TA.--;99.-.G 1.484 0.730 7255023 967 27.-.C;70.-.T 1.484 1.384 4307384 968 4.T.-;75.C.- 1.483 0.592 2702279 969 0.T.-;2.A.C;86.CC.-G 1.482 1.155 3036396 970 1.TA.--;74.-.T 1.480 0.455 10196645 971 18.-.G;78.-.C 1.479 0.758 4308690 972 4.T.-;74.-.T 1.479 0.955 4298804 973 4.T.-;78.A.G 1.477 0.725 12125860 974 2.A.-;76.G.-;78.A.C 1.476 0.787 2675530 975 0.T.-;2.A.C;90.T.- 1.474 1.266 7242260 976 27.-.C;88.G.- 1.473 1.439 4287312 977 4.T.-;82.AA.-T 1.473 0.577 3339492 978 2.A.G;0.T.-;73.AT.-C 1.472 1.445 4290113 979 4.T.-;80.A.- 1.470 0.639 2293835 980 0.T.-;78.A.-;80.A.- 1.469 0.867 6455860 981 16.-.C;74.-.C 1.468 0.527 2706303 982 0.T.-;2.C;82.AA.--;85.T.C 1.467 1.023 7252350 983 27.-.C;76.-.T 1.467 1.180 3277392 984 0.T.-;2.A.G;85.TC.-A 1.467 1.201 8538161 985 75.-.G;132.G.C 1.467 0.428 8202442 986 87.-.A 1.465 0.819 2898633 987 1.-.C;78.-.C 1.464 0.456 2648767 988 2.A.C;0.T.-;73.-.A 1.463 0.659 6115163 989 14.-.A;88.G.- 1.463 0.529 10576534 990 15.-.T;55.-.T 1.461 0.556 1904556 991 0.TTA.---;3.C.A;76.GG.-C 1.461 1.089 8073267 992 74.-.C 1.459 0.430 8755280 993 55.-.T 1.458 0.638 2341059 994 0.T.-;28.-.C 1.457 1.284 3007006 995 1.TA.--;90.T.- 1.456 1.125 7833962 996 55.-.G;87.-.G 1.456 0.883 4299868 997 4.T.-,78.-.T 1.456 0.940 8342692 998 89.A.G 1.455 0.975 2262741 999 0.T.-;85.TC.-A 1.451 0.583 1942088 1000 0.TT.--;2.A.C;86.C.- 1.450 1.216 10200245 1001 18.-.G;74.-.T 1.448 0.938 4219211 1002 4.T.-;72.-.A 1.447 0.549 2457931 1003 1.TA.--;3.C.A;75.-.C 1.444 0.736 3038631 1004 1.TA.--;73.AT.-G 1.444 0.560 12753950 1005 0.-.T;73.A.- 1.444 0.573 2129014 1006 0.TTA.---;3.C.G;75.-.G 1.440 1.366 7833901 1007 55.-.G86.C.- 1.439 0.671 10066878 1008 19.-.T;74.-.C 1.439 0.663 2714726 1009 0.T.-;2.A.C;77.GA.--;83.A.T 1.439 0.739 12106738 1010 2.A.-;72.-.G 1.438 1.201 2720418 1011 0.T.-;2.A.C;77.GA.--;80.A.C 1.436 1.201 2291924 1012 0.T.-;78.A.C 1.436 0.937 9991025 1013 19.-.G;81.GA.-T 1.434 0.688 4243954 1014 4.T.-;85.TC.-A 1.433 0.674 6362816 1015 17.-.A;75.-.C 1.433 0.887 8204227 1016 87.C.A 1.432 1.065 1980019 1017 0.T.C;78.A.- 1.431 0.702 8142815 1018 76.G.-;130.T.G 1.429 0.271 10554966 1019 15.-.T;80.A.- 1.429 1.003 2702620 1020 0.T.-;2.A.C;86.C.T 1.427 0.892 8142856 1021 76.G.-;132.G.C 1.427 0.238 12012995 1022 2.A.-.16.-.C 1.425 0.515 4284095 1023 4.T.-;82.AA.-C 1.424 0.718 10546168 1024 15.-.T;88.-.T 1.424 1.002 8128579 1025 75.-.C 1.424 0.273 2703946 1026 2.A.C.;0.T.-;82.A.-.85.T.G 1.423 1.276 12433040 1027 1.TAC.---;76.G.- 1.423 0.852 12162901 1028 2.A.-;89.-.C 1.422 0.831 2814556 1029 0.T.-;2.A.C;19.-.G 1.420 0.572 8142933 1030 76.G.-132.G.T 1.420 0.297 2710592 1031 2.A.C;0.T.-;81.-.G 1.420 0.684 8537382 1032 75.-.G;121.C.A 1.419 0.408 12434064 1033 1.TAC.---86.-.C 1.417 0.739 12438652 1034 1.TAC.---;75.C.- 1.417 0.894 8105679 1035 76.GG.-A 1.416 0.238 8089861 1036 75.-.A;86.-.C 1.414 0.397 10177945 1037 18.-.G;72.-.A 1.414 0.836 4243445 1038 4.T.-;81.GA.-C 1.413 0.887 8123491 1039 75.-.C;88.G.- 1.412 0.441 4313666 1040 4.T.-;70.-.T 1.411 0.506 7180551 1041 27.-.A;76.-.A 1.410 1.181 6534510 1042 17.-.G;76.GG.-T 1.407 0.941 3025550 1043 1.TA.--;82.AA.-T 1.407 0.570 10275000 1044 17.-.T;71.-.C 1.406 0.754 8530347 1045 75.-C.GA 1.406 0.333 12438782 1046 1.TAC.---;74.-.T 1.404 0.868 2724111 1047 2.A.C;0.T.-;78.A.-;80.A.- 1.403 1.013 12682492 1048 0.-.T;27.-.C 1.402 1.266 8336449 1049 89.-.C 1.400 0.251 2994450 1050 1.TA.--;74.-.C 1.399 0.436 10070026 1051 19.-.T;76.G.- 1.399 0.599 4246898 1052 4.T.-;86.CC.-A 1.398 0.996 2056199 1053 0.TT.--;2.A.G;82.AA.-T 1.398 1.059 2726405 1054 0.T.-;2.A.C;77.G.T 1.398 0.989 8093322 1055 75.-.A 1.396 0.309 4239175 1056 4.T.-;77.-.C 1.396 0.979 3031832 1057 1.TA.--;78.-.T 1.395 0.529 2303944 1058 0.T.-;73.A.- 1.395 0.686 2255406 1059 0.T.-;76.GG.-- 1.395 1.055 2468522 1060 1.TA.--;3.C.A;74.-.T 1.394 0.748 8543995 1061 75.-.G;86.C.- 1.393 0.372 8348831 1062 88.-.T 1.392 0.333 2899043 1063 1.-.C;78.A.- 1.392 0.693 6611143 1064 18.C.-;75.-.A 1.392 0.602 8142880 1065 76.G.- 1.391 0.256 4294538 1066 4.T.-;78.A.C 1.390 0.607 447196 1067 -27.C.A;75.-.G 1.390 0.365 3338210 1068 2.A.G;0.T.-;75.CG.-T 1.390 0.686 8538250 1069 75.-.G;131.A.C 1.389 0.442 10302419 1070 17.-.T;83.-.C 1.388 1.345 3169133 1071 0.T.-;2.A.G;16.-.C 1.388 0.627 1855234 1072 0.TT.--;86.-.C 1.387 0.590 3027053 1073 1.TA.--;80.A.- 1.386 0.444 8142905 1074 76.G.-;133.A.C 1.386 0.312 2465375 1075 1.TA.--;3.C.A;81.GA.-T 1.386 0.850 8137397 1076 76.G.-;98.-.A 1.385 0.658 3304306 1077 2.A.G;0.T.-;89.A.- 1.384 1.226 8537231 1078 75.-.G;120.C.A 1.383 0.451 4299393 1079 4.T.-;78.AG.-T 1.382 1.034 3295454 1080 2.A.G;0.T.-;99.-.G 1.382 1.039 8519489 1081 76.GG.-T 1.380 0.164 3264318 1082 2.A.G;0.T.-;75.-.A 1.379 0.703 3266116 1083 2.A.G;0.T.-;76.GG.-A 1.379 0.672 2997992 1084 1.TA.--;76.-.A 1.378 0.700 2672282 1085 2.A.C;0.T.-;86.CC.-A 1.376 0.805 14798941 1086 -29.A.C;75.-.C 1.376 0.255 12031760 1087 2.A.-;27.G.- 1.375 1.375 2201185 1088 0.T.-;16.-.C 1.373 0.446 2400173 1089 1.-.A;76.G.- 1.372 0.596 10088256 1090 19.-.T;76.G.-;78.A.T 1.370 0.715 10284913 1091 17.-.T;77.-.A 1.370 1.090 10545701 1092 15.-.T;89.A.- 1.370 1.003 8212851 1093 86.-.C 1.369 0.540 8132895 1094 75.-.C.;86.C.- 1.368 0.297 3281950 1095 2.A.G;0.T.-;86.-.C 1.368 0.907 1858655 1096 0.TT.--;87.-.G 1.368 0.620 12737396 1097 0.-.T;86.C.- 1.365 0.552 6474033 1098 16.-.C;80.A.- 1.363 0.562 2646406 1099 0.T.-;2.A.C;72.-.G 1.363 1.115 3020097 1100 1.TA.--;86.-.G 1.363 0.580 12160739 1101 2.A.-;91.A-;93.A.G 1.363 1.067 14919005 1102 -29.A.C;2.A.-;76.G.- 1.362 0.433 10527714 1103 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1.265 2279498 1128 0.T.-;88.-.T 1.350 0.488 2719218 1129 0.T.-;2.A.C;79. 1.349 1.087 GAGAAA.TTTCTC 1858516 1130 0.TT.--;86.C.- 1.349 1.337 14798574 1131 -29.A.C;76.GG-C 1..347 0.500 10178596 1132 18.-.G;72.-.C 1.346 0.766 8118222 1133 76.GG.-C;132.G.C 1.346 0.517 12181387 1134 2.A.-;82.-.T 1.345 0.639 10285141 1135 17.-.T;76.G.-78.A.C 1.345 0.980 8565359 1136 75.CG.-T 1.345 0.288 8142963 1137 76.G.-;131.A.C 1.344 0.259 6313836 1138 16.-.A;78.A.- 1.342 0.715 6455586 1139 16.-.C;74.T.- 1.341 0.589 10069022 1140 19.-.T;76.GG-C 1.339 0.689 8538125 1141 75.-.G130.T.G 1.339 0.405 8208034 1142 88.G.- 1.339 0.227 4210228 1143 4.T.-;65.G.- 1.338 0.726 8555144 1144 74.-.T;86.-.C 1.336 0.495 2211631 1145 0.T.-;27.G.- 1.336 1.023 14799468 1146 -29.A.C;76.G.- 1.335 0.265 3023524 1147 1.TA.--;82.AA.-- 1.335 0.777 14921453 1148 -29.A.C;2.A.-;75.-.G 1.334 0.448 2465666 1149 1.TA.--;3.C.A;80.A.- 1.334 1.225 2124272 1150 0.TTA.---;3.C.G;86.-.C 1.333 1.021 4366553 1151 4.T.-;28.-.C 1.333 1.147 15160651 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75.-.C;132.G.C 1.284 0.296 4026117 1202 3.-.C;76.GG.-T 1.282 0.871 3458694 1203 0.TTAC.----;75.-.C 1.282 1.236 2402393 1204 1.-.A;87.-.A 1.282 0.828 1852100 1205 0.TT.--;75.-.A 1.281 0.682 3325688 1206 2.A.G;0.T.-;78.A.- 1.281 0.892 2742029 1207 0.T.-2.A.C;73.A.T 1.281 0.548 6577492 1208 18.-.A;86.-.C 1.280 0.718 12218636 1209 2.A.-;66.CT.-G 1.279 0.773 8219007 1210 89.-.A 1.279 1.111 6369323 1211 17.-.A;76.GG.-T 1.278 0.804 2651674 1212 0.T.-;2.A.C;74.TC.-- 1.278 1.277 12717259 1213 0.-.T;74.-.C 1.277 0.541 15160113 1214 -29.A.G;76.GG.-A 1.277 0.270 2900998 1215 1.-.C;76.-.T 1.777 0.460 1864123 1216 0.TT.--;74.-.T 1.275 0.783 1936243 1217 0.TT.--;2.A.C;73.-.A 1.269 0.978 10087310 1218 19.-.T;76.-.G 1.269 1.013 8128641 1219 131.A.C;75.-.C 1.268 0.347 2466267 1220 1.TA.--;.3.C.A;78.-.C 1.268 0.761 14814370 1221 -29.A.C;74.-.T 1.268 0.225 8367586 1222 86.-.G 1.268 0.167 14814654 1223 -29.A.C;75.CG.-T 1.267 0.300 7178892 1224 27.-.A;72.-.C 1.267 1.242 2713900 1225 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1.254 0.821 10183679 1250 18.-.G;76.G.-;78.A.C 1.253 0.445 2283016 1251 0.T.-82.A.- 1.253 0.466 2695201 1252 0.T.-;2.A.C;91.A.G 1.253 0.804 6475853 1253 16.-.C;76.-.G 1.251 0.663 6111106 1254 14.-.A;76.GG.-A 1.250 0.738 3082312 1255 1.TA.--;17.-.T 1.249 0.812 10566255 1256 15.-.T;73.AT.-C 1.249 0.813 10070730 1257 19.-.T;79.G.- 1.249 0.602 14812876 1258 -29.A.C;76.GG.-T 1.248 0.151 1246999 1259 -15.T.G;76.G.- 1.247 0.225 8558498 1260 74.-.T;132.G.C 1.246 0.249 10518792 1261 15.-.T;72.-.G 1.246 0.489 4277925 1262 4.T.-;84.AT.-- 1.246 0.937 8352817 1263 86.C.- 1.245 0.151 8538048 1264 75.-.G;129.C.A 1.244 0.412 14797557 1265 -29.A.C;75.-.A 1.243 0.320 8538200 1266 75.-.G;133.A.C 1.242 0.440 4283490 1267 4.T.-;82.-.C 1.242 0.687 1865218 1268 0.TT.--;73.A.- 1.241 0.704 6525015 1269 17.-.G;75.-.A 1.241 0.979 10181717 1270 18.-.G;76.GG.-A 1.240 1.138 6458686 1271 16.-.C;76.GG.-C 1.240 0.874 9978404 1272 19.-.G;86.-.A 1.239 0.802 9631659 1273 16.------------ 1.238 1.158 .CTCATTACTTTG 1938525 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1.190 0.320 6525955 1347 17.-.G;75.-.C 1.188 1.100 6460105 1348 16.-.C;76.G.-;78.A.C 1.188 0.685 6112043 1349 14.-.A;75.-.C 1.188 0.773 1978266 1350 0.T.C;86.C.- 1.186 0.483 8636881 1351 66.CT.-G;87.-.G 1.186 0.214 15241255 1352 -29.A.G;2.A.-;75.-.G 1.186 0.444 6362433 1353 17.-.A.76.GG.-A 1.186 0.851 2059902 1354 0.TT.--;2.A.G;74.-.T 1.186 1.169 14799744 1355 -29.A.C;77.-.A 1.186 0.192 8118273 1356 76.GG.-C;132.G.T 1.185 0.630 4278865 1357 4.T.-;84.-.T 1.184 1.108 10065094 1358 19.-.T;72.-.C 1.183 0.675 8561350 1359 74.-.T;87.-.G 1.182 0.393 15160423 1360 -29.A.G;76.GG.-C 1.181 0.556 2994738 1361 1.TA.--;74.T.G 1.181 0.980 15058565 1362 -29.A.G;0.T.-;2.A.C 1.180 0.270 12222182 1363 2.A.-;65.GC.-T 1.180 0.796 2881480 1364 1.-.C;74.T.- 1.180 0.538 10193035 1365 18.-.G;86.-.G 1.178 0.685 6459089 1366 16.-.C;75.-.C 1.178 0.589 10298749 1367 17.-.T.89.-.C 1.178 0.684 8490381 1368 76.-.G;132.G.C 1.177 0.336 12106660 1369 2.A.-18.-.G 1.177 0.435 8124036 1370 75.-.C;98.-.A 1.177 0.499 2893687 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1.144 0.579 2630318 1420 2.A.C;0.T.-;66.CT.-A 1.144 0.534 1943847 1421 0.TT.--.2.A.C;81.GA.-T 1.143 0.765 4270685 1422 4.T.-;90.-.T 1.142 1.061 8066737 1423 74.T.-;131.A.C 1.142 0.298 6101577 1424 14.-.A;55.-.G 1.142 0.632 4279604 1425 4.T.-;82.A.- 1.141 0.866 2284176 1426 0.T.-;83.-.G 1.141 0.574 6480468 1427 16.-.C;70.-.T 1.140 0.614 2640116 1428 0.T.-;2.A.C;71.-.C 1.137 0.936 10194587 1429 18.-.G;82.AA.-C 1.137 0.867 15456465 1430 -30.C.G;75.-.G 1.136 0.421 3432602 1431 0.T.-;2.A.G;18.-.G 1.136 0.359 8345813 1432 89.-.T 1.135 0.634 3023247 1433 1.T.A.---83.-.T 1.135 0.960 10472698 1434 16.C.-;76.-.G 1.134 0.911 1855129 1435 0.TT.--.88.G- 1.133 0.759 9993029 1436 19.-.G;78.A.- 1.133 0.793 15168776 1437 -29.A.G;76.GG-T 1.132 0.227 2464359 1438 1.TA.--;3.C.A;82.A.-;84.A.G 1.132 1.057 12156161 1439 2.A.-;98.-.T 1.131 0.852 8544614 1440 75.-.G;82.A.- 1.131 0.458 2278784 1441 0.T.-;89.A.G 1.130 0.932 4229697 1442 4.T.-;75.CG.-A 1.129 1.031 6461360 1443 16.-.C;82.-.A 1.129 0.609 8128601 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0.444 14801930 1469 -29.A.C;88.G.- 1.115 0.262 2885740 1470 1.-.C;81.GA.-C 1.115 0.689 8436871 1471 81.GA.-T 1.115 0.274 6533591 1472 17.-.G78.-.C 1.115 0.880 8508461 1473 78.A.T 1.115 0.523 2303258 1474 0.T.-;70.-.T 1.114 0.865 10200479 1475 18.-.G;75.CG.-T 1.113 0.732 8142460 1476 76.G.-;126.C.A 1.111 0.288 8490449 1477 76.-.G;132.G.T 1.111 0.315 1862090 1478 0.TT.--;78.A.- 1.111 0.800 8105143 1479 76.GG.-A;121.C.A 1.111 0.256 10204124 1480 18.-.G;65.GC.-T 1.110 0.661 2696979 1481 0.T.-;2.A.C;88.-.G 1.110 0.607 1246393 1482 -15.T.G;76.GG.-A 1.110 0.194 4277641 1483 4.T.-;84.-.C 1.109 1.085 12163684 1484 2.A.-;88.-.G 1.109 0.570 3643882 1485 3.CT.-A;76.GG.-A 1.109 0.785 6461122 1486 16.-.C;81.GA.-C 1.108 0.626 14645694 1487 2.A.C;0.T.-;-29.A.C 1.108 0.268 2678659 1488 0.T.-;2.A.C;98.-.A 1.108 0.376 2295085 1489 0.T.-;77.GA.--;80.A.T 1.108 0.695 8127785 1490 75.-.C;120.C.A 1.107 0.299 8357871 1491 87.-.G;132.G.T 1.107 0.336 12090020 1492 2.A.-;66.CT.-A 1.106 0.760 3079463 1493 1.TA.--;19.-.T 1.105 0.424 10277558 1494 17.-.T;72.-.G 1.105 0.335 2694724 1495 0.T.-;2.A.C;92.A.T 1.102 0.929 3135565 1496 1.T.G;3.C.-;75.C.- 1.102 0.673 6304328 1497 16.-.A;75.-.C 1.102 0.655 2708067 1498 2.A.C;0.T.-;83.-.T 1.102 0.859 6469331 1499 16.-.C;89.A.- 1.101 0.791 10073526 1500 19.-.T;90.T.- 1.101 0.917 3017595 1501 1.TA.--;89.AT.-G 1.101 0.904 3031194 1502 1.TA.--;78.A.G 1.100 1.042 12123777 1503 2.A.-;76.G.-;132.G.C 1.100 0.426 15451300 1504 -30.C.G;76.G.- 1.100 0.258 8105041 1505 76.GG.-A;120.C.A 1.100 0.198 2894267 1506 1.-.C;87.-.T 1.099 0.722 2998547 1507 1.TA.--;76.GG.-C 1.099 0.772 3022051 1508 1.TA.--;83.-.C 1.099 0.800 8512487 1509 76.G.-;78.A.T 1.098 0.434 2285757 1510 0.T.-;82.AA.-C 1.098 0.581 6531470 1511 17.-.G;87.-.G 1.097 0.892 3461447 1512 0.TTAC.----;78.A.- 1.097 1.032 6475031 1513 16.-.C;78.-.C 1.096 0.623 10194914 1514 18.-.G;82.AA.-G 1.095 0.926 1041972 1515 -17.C.A;76.G.- 1.094 0.260 8537811 1516 75.-.G;126.C.A. 1.094 0.416 3020817 1517 1.TA.--;84.AT.-- 1.094 1.006 2887379 1518 1.-.C;86.-.C 1.093 0.650 1854285 1519 0.TT.--;77.GA.-- 1.093 0.836 8357326 1520 87.-.G;121.C.A 1.093 0.228 8128534 1521 75.-.C;130.T.G 1.092 0.292 1947291 1522 0.TT.--;2.A.C;73.A.- 1.092 1.083 12432721 1523 1.TAC.---;76.GG.-C 1.091 0.425 1252779 1524 -15.T.G;75.-.G 1.091 0.436 3588353 1525 2.-.A;86.-.C 1.090 0.473 2900664 1526 1.-.C;76.GG.-T 1.090 0.928 8076983 1527 74.T.G 1.090 0.516 2300899 1528 0.T.-;73.-.C 1.088 0.922 12202788 1529 2.A.-;75.-.G;132.G.C 1.087 0.397 10070325 1530 19.-.T;77.-.A 1.085 0.602 14685826 1531 -29.A.C;4.T.-;76.G.- 1.085 0.875 14351033 1532 -25.A.C;75.-.G 1.085 0.402 8607376 1533 73.A.T 1.084 0.466 12439360 1534 1.TAC.---;73.A.- 1.084 0.785 12718596 1535 0.-.T;75.-.A 1.083 0.730 2712801 1536 2.A.C;0.T.-;82.A.T 1.083 1.030 6613293 1537 18.C.-;77.-.C 1.082 0.704 8480766 1538 78.A.- 1.081 0.244 2414074 1539 1.-.A;75.CG.-T 1.078 0.690 8105662 1540 76.GG.-A;132.G.C 1.078 0.266 2282078 1541 0.T.-;84.AT.-- 1.078 1.018 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76.-.G;87.-.G 1.065 0.356 8148054 1567 76.G.-;87.-.G 1.065 0.414 2684598 1568 0.T.-;2.A.C;133.A.C 1.064 0.264 1806606 1569 -3.TAGT.----;76.G.- 1.063 0.955 6112609 1570 14.-.A;76.G.- 1.063 0.690 8128619 1571 75.-.C;132.G.T 1.063 0.341 2263869 1572 0.T.-;85.-.G 1.062 1.017 8519538 1573 76.GG.-.T;131.A.C 1.061 0.210 15167837 1574 -29.A.G;78.A.- 1.061 0.247 8539891 1575 113.A.C;75.-.G 1.061 0.380 6110621 1576 14.-.A;75.-.A 1.060 0.621 4012102 1577 3.-.C;76.GG.-A 1.059 1.032 14644765 1578 -29.A.C;0.T.-;2.A.C;76.GG.-A 1.059 0.330 6114928 1579 14.-.A;87.-.A 1.058 0.886 1858781 1580 0.TT.--;87.-.T 1.058 0.825 10090936 1581 19.-.T;75.CG.-T 1.056 0.659 2002673 1582 0.TTA.---;86.-.C 1.055 0.913 1937274 1583 0.TT.--;2.A.C;76.-.A 1.055 0.766 1946930 1584 2.A.C;0.TT.--;73.AT.-G 1.054 1.042 8564806 1585 75.CG.-T;121.C.A 1.054 0.274 14646874 1586 -29.A.C;0.T.-;2.A.C;78.A.- 1.053 0.595 3279449 1587 2.A.G;0.T.-;86.-.A 1.053 0.589 10183929 1588 18.-.G;79.G.- 1.052 0.658 4281239 1589 4.T.-;83.-.G 1.052 0.864 8636987 1590 66.CT.-G;87.-.T 1.052 0.463 2684414 1591 129.C.A;2.A.C;0.T.- 1.051 0.312 10567800 1592 15.-.T;70.-.T 1.050 0.621 12183487 1593 2.A.-;77.GA.--;83.A.T 1.049 0.987 3429655 1594 0.T.-;2.A.G;19.-.T 1.049 0.495 15168064 1595 -29.A.G;76.-.G 1.048 0.302 8579268 1596 73.A.C 1.048 0.683 12725378 1597 0.-.T;86.-.A 1.047 0.366 12133179 1598 2.A.-;85.TC.-- 1.047 0.820 12169171 1599 2.A.-;87.C.T 1.047 0.600 1974530 1600 0.T.C;74.-.G 1.045 0.682 3276852 1601 2.A.G;0.T.-;81.GA.-C 1.045 0.975 2277126 1602 0.T.-;91.A.-;93.A.G 1.044 0.955 2668148 1603 0.T.-;2.A.C;80.-.A 1.043 0.586 1946365 1604 0.TT.--;2.A.C;74.-.T 1.043 1.041 10086224 1605 19.-.T;78.AG.-C 1.043 0.736 6474902 1606 16.-.C;78.AG.-C 1.042 0.503 3001790 1607 1.TA.--;77.-.C 1.042 0.684 6463023 1608 16.-.C;89.-.A 1.042 0.830 8470293 1609 78.-.C;132.G.T 1.042 0.300 3134206 1610 1.T.G;3.C.- 1.041 0.793 10203551 1611 18.-.G;66.CT.-G 1.040 0.787 8629503 1612 66.CT.-G;86.-.C 1.039 0.370 13846013 1613 -14.A.C;76.G.- 1.038 0.247 2263715 1614 0.T.-;85.TC.-G 1.038 0.802 10560681 1615 15.-.T;78.A.T 1.038 0.677 1253221 1616 -15.T.G;75.CG-T 1.038 0.213 10556907 1617 15.-.T;78.AG.-C 1.037 1.020 3319204 1618 0.T.-2.A.G;77.GA.--;83.A.T 1.036 0.978 2277677 1619 0.T.-;91.AA.-G 1.035 0.945 3044097 1620 1.TA.--;65.GC.-T 1.034 0.777 2728986 1621 0.T.-;2.A.C;76.GG.--;78.A.T 1.033 0.961 15059527 1622 -29.A.G;0.T.-2.A.C;75.-.G 1.033 0.531 8127925 1623 75.-.C;121.C.A 1.032 0.246 8069875 1624 74.T.-;87.-.G 1.032 0.583 4210905 1625 4.T.-;66.CT.-A 1.032 0.842 393375 1626 -27.C.A;0.T.-2..A.C 1.031 0.249 6469193 1627 16.-.C;88.-.G 1.030 0.736 12723788 1628 0.-.T;77.GA.-- 1.030 0.436 1975104 1629 0.T.C;75.-.C 1.030 0.579 447486 1630 -27.C.A;74.-.T 1.030 0.222 2304326 1631 0.T.-;73.A.T 1.029 0.531 8480805 1632 78.A.-;132.G.T 1.029 0.245 10289207 1633 17.-.T;89.-.A 1.026 0.760 10541758 1634 15.-.T;99.-.G 1.026 0.736 8580639 1635 73.-TC.G- 1.026 0.359 2129400 1636 0.TTA.---;3.C.G;74.-.T 1.026 1.011 8142671 1637 76.G.-128.T.G 1.026 0.290 12726231 1638 0.-.T;88.G.- 1.026 0.405 10288957 1639 17.-.T;88.GA.-C 1.025 0.602 2982939 1640 1.TA.--;65.GC.-A 1.025 0.854 8357852 1641 87.-.G;133.A.C 1.024 0.267 6626305 1642 18.C.-;76.-.G 1.024 0.941 15167605 1643 -29.A.G;78.-.C 1.024 0.228 3273923 1644 2.A.G;0.T.-;79.G.- 1.022 0.761 10553626 1645 15.T;82.AA.-T 1.020 0.844 3029129 1646 1.TA.--;78.A.C 1.018 0.493 3133667 1647 1.T.G;3.C.-;76.G.- 1.018 0.664 14921066 1648 -29.A.C;2.A.-;78.A.- 1.018 0.654 14806598 1649 -29.A.C;88-T 1.017 0.327 8139512 1650 115.T.G;76.G.- 1.017 0.260 8636794 1651 66.CT.-G;86.C.- 1.017 0.224 8127584 1652 75.-.C;119.C.A 1.017 0.258 4311933 1653 4.T.-;73.-.G 1.016 0.722 6471359 1654 16.-.C;83.-.C 1.016 0.690 12433542 1655 1.TAC.--;77.GA.- 1.015 0.963 8093303 1656 75.-.A;132.G.C 1.014 0.287 1246761 1657 -15.T.G;75.-.C 1.014 0.245 1943763 1658 0.TT.--;2.A.C;82.AA.-T 1.013 0.876 4158980 1659 4.T.-;16.-.C 1.012 0.731 8470306 1660 78.-.C;131.A.C 1.012 0.269 8069089 1661 74.T.-;98.-.T 1.012 0.754 12438882 1662 1.TAC.---;75.CG.-T 1.012 0.646 8338521 1663 89.AT.-G 1.010 0.922 10088951 1664 19.-.T;76.-.T 1.010 0.995 12163085 1665 2.A.-;89.A.C 1.010 1.006 8479927 1666 78.A.-;121.C.A 1.008 0.198 10196772 1667 18.-.G;78.A.C 1.007 0.606 8552295 1668 75.C.-;87.-.G 1.006 0.446 4027916 1669 3.-.C;74.-.T 1.006 0.888 8489338 1670 76.-.G;119.C.A 1.005 0.338 446968 1671 -27.C.A;76.GG.-T 1.005 0.187 2049927 1672 0.TT.--;2.A.G;88.G.- 1.005 0.953 8598621 1673 70.-.T;87.-.G 1.004 0.383 8600573 1674 73.A.-;86.-.C 1.004 0.369 8473900 1675 78.A.C 1.003 0.272 12174360 1676 2.A.-;83.-.C 1.002 0.612 442458 1677 -27.C.A;76.G.- 1.001 0.255 15162537 1678 -29.A.G;86.-.C 1.000 0.512 2991036 1679 1.TA.--;72.-.C 0.999 0.524 8489557 1680 76.-.G;120.C.A 0.999 0.235 2704195 1681 0.T.-;2.A.C;84.A.G 0.999 0.779 12746931 1682 0.-.T;78.AG.-T 0.999 0.695 8544289 1683 75.-.G;86.-.G 0.998 0.330 8490052 1684 76.-.G;126.C.A 0.998 0.284 3003857 1685 1.TA.--;81.GA.-C 0.997 0.622 2683589 1686 0.T.-;2.A.C;121.C.A 0.997 0.259 8565256 1687 75.CG.-T;129.C.A 0.996 0.264 2684649 1688 0.T.-;2.A.C;131.A.C 0.995 0.272 10192242 1689 18.-.G;88.-.T 0.995 0.989 8128468 1690 75.-.C;129.C.A 0.995 0.262 3255338 1691 2.A.G;0.T.-;72.-.C 0.994 0.842 7829410 1692 55.-.G;75.-.C 0.994 0.860 15162331 1693 -29.A.G;87.-.A 0.993 0.691 8212834 1694 86.-.C;132.G.C 0.992 0.467 13222300 1695 2.A.G;-3.TAGT----;76.G.- 0.991 0.723 8470255 1696 78.-.C;132.G.C 0.991 0.219 2661937 1697 132.G.C;2.A.C;0.T.-;76.G.- 0.990 0.390 2670761 1698 0.T.-;2.A.C;85.TCC.-- 0.990 0.720 11776916 1699 2.-.C;87.-.A 0.989 0.938 12747759 1700 0.-.T;77.-.T 0.989 0.938 15165085 1701 -29.A.G;86.C.- 0.987 0.176 8212745 1702 86.-.C;129.C.A 0.987 0.509 2989789 1703 1.TA.--;72.-.A 0.986 0.659 6531564 1704 17.-.G;87.-.T 0.985 0.962 12436169 1705 1.TAC.---;87.-.G 0.984 0.678 3311127 1706 2.A.G;0.T.-;82.A.- 0.984 0.759 2264270 1707 0.T.-;86.CC.-A 0.983 0.775 10091719 1708 19.-.T;73.AT.-G 0.982 0.402 8143233 1709 76.G.-;123.A.C 0.982 0.226 1248077 1710 -15.T.G;86.-.C 0.981 0.619 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8142576 1735 76.G.-;127.T.G 0.973 0.375 14816291 1736 -29.A.C;73.A.- 0.972 0.232 10080185 1737 19.-.T;89.-.C 0.971 0.565 1904247 1738 0.TTA.---;3.C.A;75.-.A 0.970 0.749 6460821 1739 16.-.C;77.GA.-- 0.970 0.637 12738126 1740 0.-.T;87.-.T 0.968 0.578 8357730 1741 87.-.G;129.C.A 0.968 0.270 12187919 1742 2.A.-;79.GA.-T 0.968 0.963 14644862 1743 -29.A.C;0.T.-;2.A.C;76.GG.-C 0.967 0.512 13101334 1744 -1.GT.--;76.GG.-T 0.967 0.377 12437308 1745 1.TAC.---;80.A.- 0.966 0.933 2672055 1746 0.T.-;2.A.C;86.C.A 0.966 0.590 6304109 1747 16.-.A;76.GG.-C 0.966 0.672 12214091 1748 2.A.-;73.A.T 0.966 0.602 8511126 1749 76.G.-;78.AG.TC 0.965 0.454 10473646 1750 16.C.-;76.GG.-T 0.965 0.499 8561622 1751 74.-.T;82.A.- 0.965 0.362 1981516 1752 0.T.C;75.C.- 0.964 0.575 4300894 1753 4.T.-;77.G.T 0.964 0.236 8084158 1754 74.-.G 0.964 0.402 8096194 1755 75.-.A;87.-.T 0.964 0.605 2281085 1756 0.T.-;87.C.T 0.961 0.675 8063355 1757 74.T.-;86.-.C 0.960 0.507 3038327 1758 1.TA.--;73.-.G 0.959 0.854 9976817 1759 19.-.G;79.G.- 0.958 0.737 13223005 1760 2.A.G;-3.TAGT.---- 0.958 0.837 8542589 1761 75.-.G;98.-.T 0.957 0.875 3345006 1762 0.T.-;2.A.G;73.A.T 0.957 0.793 4217628 1763 4.T.-;71.-.C 0.956 0.495 10068711 1764 19.-.T;76.-.A 0.956 0.689 10198139 1765 18.-.G;77.-.T 0.956 0.663 2463484 1766 1.TA.--;3.C.A;87.-.T 0.955 0.695 8490228 1767 76.-.G;128.T.G 0.955 0.305 3322121 1768 0.T.-;2.A.G;80.AG.-T 0.955 0.812 2458850 1769 1.TA.--;3.C.A;79.G.- 0.955 0.858 6626017 1770 18.C.-;78.A.- 0.954 0.611 8519520 1771 76.GG.-T;132.G.T 0.954 0.281 1974653 1772 0.T.C;75.-.A 0.954 0.490 2683428 1773 120.C.A;2.A.C;0.T.- 0.954 0.253 4272200 1774 4.T.-;89.A.G 0.954 0.925 8193481 1775 85.TC.-G 0.953 0.701 6557686 1776 18.C.A;75.-.G 0.953 0.330 1860902 1777 0.TT.--;81.GA.-T 0.952 0.515 2717874 1778 2.A.C;0.T.-;80.AG.-T 0.951 0.611 2882024 1779 1.-.C;74.-.G 0.951 0.619 3273132 1780 0.T.-;2.A.G;77.-.C 0.951 0.397 441958 1781 -27.C.A;76.GG.-A 0.949 0.205 14811390 1782 -29.A.C;78.A.- 0.949 0.249 14802094 1783 -29.A.C;86.-.C 0.949 0.461 10523926 1784 15.-.T;76.-.A 0.948 0.739 12742835 1785 0.-.T;81.GA.-T 0.948 0.383 8093342 1786 75.-.A;133.A.C 0.948 0.377 8490265 1787 76.-.G;129.C.A 0.948 0.322 2412848 1788 1.-.A;76.-.T 0.947 0.632 8183422 1789 85.TC.-A 0.947 0.638 2463159 1790 1.TA.--;3.C.A;88.-.T 0.946 0.552 8490433 1791 76.-.G;133.A.C 0.946 0.318 2681222 1792 0.T.-;2.A.C;115.T.G 0.946 0.288 8480741 1793 78.A.-;132.G.C 0.946 0.202 2663534 1794 0.T.-;2.A.C;77.G.C 0.946 0.861 8118132 1795 76.GG.-C;129.C.A 0.946 0.373 6447398 1796 16.-.C;55.-.G 0.945 0.768 2285156 1797 0.T.-;82.AA.-- 0.945 0.503 8117520 1798 76.GG.-C;120.C.A 0.945 0.413 8603147 1799 73.A.- 0.945 0.225 8537609 1800 75.-.G;124.T.G 0.944 0.366 2245955 1801 0.T.-;71.-.C 0.944 0.684 8161116 1802 79.G.- 0.942 0.264 8536998 1803 75.-.G;119.C.A 9.942 0.370 8537871 1804 75.-.G;127.T.C 0.941 0.334 8543767 1805 75.-.G;89.A.- 0.941 0.628 6603080 1806 18.C.-;55.-.G 0.941 0.707 13850293 1807 -14.A.C;87.-.G 0.940 0.218 1852615 1808 0.TT.--;76.-.A 0.938 0.750 8208020 1809 88.G.-;132.G.C 0.938 0.242 14918769 1810 -29.A.C;2.A.-;76.GG.-A 0.937 0.353 8223161 1811 90.-.G 0.937 0.664 2684123 1812 0.T.-;2.A.C;126.C.A 0.936 0.262 2883487 1813 1.-.C;76.GG.-C 0.934 0.884 8089075 1814 75.-C.AA 0.934 0.299 13746840 1815 -13.G.T;76.G- 0.934 0.266 10179608 1816 18.-.G;73.-.A 0.933 0.587 8357113 1817 87.-.G;119.C.A 0.933 0.238 2570963 1818 0.T.-;2.A.C;18.C.- 0.932 0.404 6621548 1819 18.C.-88.-.T 0.932 0.702 8543544 1820 75.-.G;89.-.C 0.930 0.331 8158269 1821 79.G.A 0.928 0.860 3341556 1822 2.A.G;0.T.-;73.AT.-G 0.928 0.857 2683151 1823 119.C.A;2.A.C;0.T.- 0.928 0.288 8543919 1824 75.-.G;88.-.T 0.926 0.543 2570189 1825 0.T.-;2.A.C;18.-.A 0.926 0.645 4015474 1826 3.-.C;86.-.C 0.926 0.838 2731496 1827 0.T.-;2.A.C;75.-.G;132.G.C 0.925 0.518 8480834 1828 78.A.-;131.A.C 0.925 0.257 3011827 1829 1.TA.-- 0.923 0.388 8592843 1830 70.-.T;86.-.C 0.923 0.501 8057655 1831 73.-.A 0.923 0.547 8480787 1832 78.A.-;133.A.C 0.923 0.247 2249456 1833 0.T.-;72.-.G 0.922 0.820 8752628 1834 55.-.T;76.GG.-A 0.922 0.503 2274200 1835 0T.-;99.-.T 0.921 0.848 8142972 1836 76.G.-;131.A.C;133.A.C 0.921 0.258 1252489 1837 -15.T.G;76.GG.-T 0.921 0.236 14822468 1838 -29.A.C;55.-.T 0.921 0.524 8357890 1839 87.-.G;131.A.C 0.921 0.275 8485265 1849 76.-.G;88.G.- 0.920 0.453 14796763 1841 -29.A.C;74.-.C 0.919 0.375 14796493 1842 -29.A.C;74.T.- 0.919 0.249 8558538 1843 74.-.T;133.A.C 0.919 0.281 7247803 1844 27.-.C;86.CC.-G 0.918 0.915 10073442 1845 19.-.T;88.GA.-C 0.918 0.552 12133660 1846 2.A.-;85.TC.-G 9.918 0.916 2572420 1847 0.T.-;2.A.C.;19.-.A 0.917 0.558 8555076 1848 74.-.T;88.G.- 0.915 0.377 19607377 1849 16.C.T;75.-.G 0.915 0.789 3281290 1850 2.A.G;0.T.-;88.G.- 0.915 0.699 12713711 1851 0.-.T;72.-.A 0.915 0.659 15408234 1852 -30.C.G;0.T.-;2.A.C 0.915 0.291 12722990 1853 0.-.T;79.G.- 0.915 0.499 8105716 1854 76.GG.-A;132.G.T 0.914 0.275 2271180 1855 0.T.- 9.913 0.381 10289412 1856 17.-.T;90.-.G 0.913 0.695 14807090 1857 -29.A.C.;87.-.T 0.912 0.449 6108421 1858 14.-A;72.-.C 0.910 0.863 8141461 1859 76.G.-;119.C.A 0.909 0.263 14350324 1860 -25.A.C;76.-.G 0.908 0.330 8538185 1861 130.--T.TAG;133.A.G;75.-.G 0.906 0.421 8538491 1862 75.-.G;123.A.C 0.906 0.359 14292135 1863 -25.A.C;0.T.-;2.A.C 0.905 0.255 2399779 1864 1.-.A;75.-.C 0.904 0.626 8142947 1865 76.G.-;131.AG.CC 0.903 0.312 8603195 1866 73.A.-;131.A.C 0.902 0.229 3329015 1867 2.A.G;0.T.-;78.-.T 0.901 0.635 2457498 1868 1.TA.--;3.C.A;76.-.A 0.901 0.878 14799938 1869 -29.A.C;76.G.-;78.A.C 0.901 0.250 10194359 1870 18.-.G-;82.AA.-- 0.901 0.723 2461767 1871 1.TA.--;3.C.A;99.-.G 0.898 0.891 8128631 1872 75.-.C;131.AG.CC 0.898 0.298 6130904 1873 14.-.A-75.CG.-T 0.898 0.809 2885480 1874 1.-.C;77.GA.-- 0.897 0.564 8565409 1875 131.A.C;75.CG.-T 0.896 0.289 8526599 1876 76.-.T;133.A.C 0.895 0.367 8542268 1877 75.-.G;99.-.G 0.895 0.466 3296935 1878 0.T.-;2.A.G;98.-.T 0.894 0.819 8535676 1879 115.T.G;75.-.G 0.892 0.386 8530925 1880 75.-.G;82.-.A 0.891 0.434 8142901 1881 76.G.-;134.G.T 0.890 0.290 8142383 1882 76.G.-;125.T.G 0.890 0.343 2054253 1883 0.TT.--;2.A.G;87.-.T 0.890 0.872 8001281 1884 71.T.C 0.888 0.608 6366788 1885 17.-.A;86.C.- 0.888 0.797 12123821 1886 2.A.-;76.G.-;131.A.C 0.887 0.303 15159066 1887 -29.A.G;74.T.- 0.886 0.228 10072842 1888 19.-.T;87.-.A 0.886 0.612 1979426 1889 0.T.C;80.A.- 0.886 0.576 10193667 1890 18.-.G;82.A.- 0.886 0.828 1252039 1891 -15.T.G;76.-.G 0.885 0.316 4247573 1892 4.T.-;87.C.A 0.885 0.526 6110295 1893 14.-.A;74.-.G 0.884 0.833 6369429 1894 17.-.A;76.-.T 0.884 0.672 6476407 1895 16.-.C;78.-.T 0.883 0.612 2309043 1896 0.T.-;65.GC.-T 0.883 0.649 10084280 1897 19.-.T;82.AA.-G 0.883 0.750 2884850 1898 1.-.C;76.G.-;78.A.C 0.882 0.492 2347258 1899 0.T.-;19.-.G 0.880 0.616 12737110 1900 0.-.T;88.-.T 0.880 0.357 10557558 1901 15.-.T;78.A.C 0.879 0.710 1851901 1902 0.TT.--;74.-.G 0.878 0.824 6621723 1903 18.C.-;86.C.- 0.877 0.845 10567449 1904 15.-.T;73.A.G 0.876 0.489 1863878 1905 0.TT.--;75.C.- 0.876 0.766 7832261 1906 55.-.G;132.G.C 0.876 0.807 15161180 1907 -29.A.G;77.-.A 0.875 0.216 8545164 1908 75.-.G;82.AA.-G 0.875 0.569 7830386 1909 55.-.G;86.-.C 0.875 0.744 6077749 1910 15.TC.-A;76.G.- 0.875 0.859 8148008 1911 76.G.-;86.C.- 0.875 0.187 2278635 1912 0.T.-;88.-.G 0.874 0.725 1041817 1913 -17.C.A;75.-.C 0.873 0.246 2465231 1914 1.TA.--;3.C.A;82.AA.-T 0.873 0.830 2266703 1915 0.T.-;90.-.G 0.872 0.862 6625678 1916 18.C.-;78.-.C 0.872 0.580 8136927 1917 76.G.-;86.-.C 0.872 0.493 8093375 1918 75.-.A;131.A.C 0.871 0.335 2454809 1919 1.TA.--;3.C.A;72.-.A 0.870 0.736 1980576 1920 0.T.C;76.GG-T 0.870 0.466 2271158 1921 0.T.-;132.G.C 0.870 0.383 442251 1922 -27.C.A;75.-.C 0.870 0.273 2350399 1923 0.T.-;18.-.G 0.869 0.556 8498008 1924 78.A.G 0.869 0.356 8080600 1925 74.-.G;86.-.C 0.868 0.560 3328595 1926 2.A.G;0.T.-;78.AG.-T 0.868 0.824 8467079 1927 78.AG.-C 0.868 0.422 6459918 1928 16.-.C;77.-.A 0.866 0.523 2265855 1929 0.T.-;88.GA.-C 0.865 0.721 15161451 1930 -29.A.G;79.G.- 0.865 0.291 8565376 1931 75.CG.-T;133.A.C 0.865 0.308 2684676 1932 0.T.-;2.A.C;131.A.G 0.864 0.347 6461858 1933 16.-.C;86.-.A 0.864 0.611 3011807 1934 1.TA.--;132.G.C 0.863 0.396 1905700 1935 0.TTA.---;3.C.A;86.-.C 0.863 0.792 8440297 1936 81.GAA.-TT 0.863 0.410 8752800 1937 55.-.T;75.-.C 0.862 0.546 12721020 1938 0.-.T;75.-.C 0.862 0.449 441780 1939 -27.C.A;75.-.A 0.861 0.300 10070497 1940 19.-.T;76.G.-;78.A.C 0.861 0.561 8112403 1941 76.-.A;132.G.T 0.861 0.584 1002534 1942 -17.C.A;2.A.C;0.T.- 0.861 0.277 3324612 1943 0.T.-;2.A.G;78.A.C 0.861 0.737 3030912 1944 1.TA.--;78.A.-;80.A.- 0.861 0.838 10182195 1945 18.-.G;76.GG.-C 0.860 0.462 8519380 1946 76.GG.-T;129.C.A. 0.860 0.207 8493521 1947 76.-.G;98.-.T 0.859 0.735 8128428 1948 75.-.C;128.T.G 0.858 0.241 1248006 1949 -15.T.G;88.G.- 0.857 0.217 5585921 1950 10.T.C;76.G.- 0.855 0.371 6127219 1951 14.-.A;78.A.- 0.855 0.493 3007558 1952 1.TA.--;90.-.G 0.854 0.711 10555821 1953 15.-.T;80.AG.-T 0.854 0.843 12747339 1954 0.-.T;78.A.T 0.854 0.745 14344892 1955 -25.A.C;75.-.C 0.853 0.296 10310038 1956 17.-.T;77.-.T 0.853 0.647 4303315 1957 4.T.-;76.G.T 0.852 0.664 14786751 1958 -29.A.C;55.-.G 0.851 0.737 15050318 1959 -29.A.G;0.T.-;2.A.C;76.-.G 0.851 0.285 15240190 1960 -29.A.G;2.A.- 0.851 0.500 6468525 1961 16.-.C;91.A.-;93.A.G 0.849 0.652 2826831 1962 0.T.-;2.A.C;15.-.T;75.-.G 0.849 0.523 8212871 1963 86.-.C;133.A.C 0.848 0.669 3318144 1964 2.A.G;0.T.-;82.AA.-T 0.848 0.742 1246180 1965 -15.T.G;75.-.A 0.847 0.337 1982591 1966 0.T.C;66.CT.-G 0.847 0.442 15166880 1967 -29.A.G;81.GA.-T 0.847 0.253 1904171 1968 0.TTA.---;3.C.A;74.-.G 0.846 0.783 14635061 1969 -29.A.C;0.T.- 0.846 0.382 8565091 1970 75.CG.-T;126.C.A. 0.845 0.207 2725821 1971 0.T.-;2.A.C;77.GA.--;80.A.T 0.845 0.837 4259960 1972 4.T.-;130.T.G 0.844 0.800 3135495 1973 1.T.G;3.C.-;75.-.G 0.844 0.791 14345120 1974 -25.A.C;76.G.- 0.844 0.259 10071193 1975 19.-.T;81.G.- 0.844 0.779 6476304 1976 16.-.C;78.AG.-T 0.844 0.661 15175052 1977 -29.A.G;55.-.T 0.844 0.629 8519203 1978 76.GG.-T;126.C.A 0.843 0.233 8173991 1979 77.GA.-- 0.843 0.383 12746208 1980 0.-.T;76.-.G 0.842 0.435 8133056 1981 75.-.C;87.-.T 0.842 0.419 8526626 1982 76.-.T;131.A.C 0.841 0.223 1252968 1983 -15.T.G;75.C.- 0.841 0.361 14646713 1984 -29.A.C;0.T.-;2.A.C;80.A.- 0.840 0.513 6304778 1985 16.-.A;77.-.A 0.840 0.462 8479746 1986 78.A.-120.C.A 0.838 0.293 12763666 1987 0.-.T;55.-.T 0.838 0.783 2684656 1988 0.T.-;2.A.C;131.A.C;133.A.C 0.838 0.207 14800177 1989 -29.A.C;79.G.- 0.837 0.233 8128118 1990 75.-.C.124.T.G 0.837 0.256 13797685 1991 -14.A.C;0.T.-;2.A.C 0.836 0.250 4259801 1992 4.T.-;128.T.G 0.836 0.763 6612829 1993 18.C.-;76.G.- 0.833 0.708 448172 1994 -27.C.A;73.A.- 0.833 0.216 1246589 1995 -15.T.G;76.GG.-C 0.833 0.560 14796144 1996 -29.A.C;73.-.A 0.832 0.441 6611642 1997 18.C.-;76.GG.-A 0.831 0.704 3040392 1998 1.TA.--;73.A.T 0.831 0.517 1938331 1999 0.TT.--;2.A.C;79.G.- 0.831 0.783 10528065 2000 15.-.T;79.GA.-C 0.831 0.713 3261986 2001 0.T.-;2.A.G;74.T.G 0.830 0.736 8131593 2002 75.-.C;99.-.G 0.830 0.553 14255597 2003 -24.G.T;2.A.- 0.830 0.570 14879001 2004 -29.A.C;15.-.T;75.-.G 0.829 0.805 14918841 2005 -29.A.C;2.A.-;76.GG.-C 0.829 0.732 2290589 2006 0.T.-;79.GA.-T 0.829 0.726 2951795 2007 1.TA.--;16.-.C 0.829 0.306 9987799 2008 19.-.G;86.-.G 0.827 0.731 15455726 2009 -30.C.G;78.A.- 0.827 0.282 11812695 2010 -29.A.C;77.-.T 0.826 0.575 8202480 2011 87.-.A;131.A.C 0.825 0.570 8066107 2012 74.T.-121.C.A 0.825 0.204 14807234 2013 -29.A.C;86.-.G 0.824 0.174 10085211 2014 19.-.T;80.A.- 0.824 0.633 8180233 2015 81.GA.-C 0.873 0.428 1044371 2016 -17.C.A;87.-.G 0.821 0.293 10286908 2017 17.-.T;85.TC.-A 0.821 0.502 10250881 2018 18.C.T;75.-.G 0.820 0.593 2463586 2019 1.TA.--;3.C.A;86.-.G 0.820 0.682 6554412 2020 18.C.A;76.G.- 0.819 0.318 8485725 2021 76.-.G;98.-.A 0.818 0.716 2271237 2022 0.T.-;131.A.C 0.817 0.352 2564816 2023 0.T.-;2.A.C;17.-.A 0.816 0.601 8357229 2024 87.-.G;120.C.A 0.816 0.329 12747630 2025 0.-.T;76.G.-;78.A.T 0.816 0.796 9972115 2026 19.-.G;73.-.A 0.816 0.802 8212329 2027 86.-.C;121.C.A 0.815 0.514 14654311 2028 -29.A.C;1.TA.--;76.G.- 0.815 0.380 1864798 2029 0.TT.--;73.AT.-G 0.814 0.762 8117352 2030 76.GG.-C;119.C.A 0.813 0.433 8479512 2031 78.A.-;119.C.A 0.812 0.224 8133372 2032 75.-.C;82.A.- 0.812 0.357 10468894 2033 16.C.-;87.-.G 0.812 0.667 8489702 2034 76.-.G;121.C.A 0.812 0.335 14919783 2035 -29.A.C;2.A.- 0.812 0.513 8198335 2036 86.C.A. 0.811 0.799 8105698 2037 76.GG.-A;133.A.C 0.811 0.269 13845556 2038 -14.A.C;76.GG.-C 0.809 0.491 3011864 2039 1.TA.--;132.G.T 0.809 0.352 13222066 2040 2.A.G;-3.TAGT.----;76.GG.-A 0.809 0.597 6471171 2041 16.-.C;82.A.- 0.808 0.510 8526572 2042 132.G.C;76.-.T 0.808 0.259 8352868 2043 86.C.-;131.A.C 0.807 0.226 10198068 2044 18.-.G;76.G.-;78.A.T 0.807 0.436 8137025 2045 76.G.-;89.-.A 0.804 0.538 8629413 2046 66.CT.-G;88.G.- 0.803 0.320 8105428 2047 76.GG.-A;126.C.A 0.803 0.240 7947397 2048 66.CT.-A.87.-.G 0.802 0.362 7835793 2049 55.-.G;76.GG.-T 0.802 0.735 8140338 2050 76.G.-;116.T.G 0.802 0.306 12722736 2051 0.-.T;77.-.C 0.801 0.427 8757065 2052 55.-.T;86.C.- 0.801 0.559 2398681 2053 1.-.A;75.-.A 0.801 0.641 4011043 2054 3.-.C;74.-.C 0.799 0.713 14920334 2055 -29.A.C;2.A.-;86.C.- 0.799 0.460 13845318 2056 -14.A.C;76.GG-A 0.799 0.188 3427589 2057 0.T.-;2.A.G;19.-.G 0.799 0.416 14806422 2058 -29.A.C.;89.A.- 0.798 0.702 15165304 2059 -29.A.G;87.-.T 0.797 0.463 2125941 2060 0.TTA.---;3.C.G;89.A.- 0.797 0.791 15168973 2061 -29.A.G;76.-.T 0.796 0.380 8538239 2062 75.-.G;131.AG.CC 0.796 0.429 8528721 2063 76.GGA.-TT 0.796 0.447 7834109 2064 55.-.G;86.-.G 0.794 0.596 8476335 2065 78.A.-;98.-.A 0.794 0.528 8352802 2066 132.G.C;86.C.- 0.794 0.214 10372832 2067 18.CA.-T;74.-.T 0.794 0.724 8752727 2068 55.-.T;76.GG.-C 0.793 0.681 6460172 2069 16.-.C;77.-.C 0.792 0.474 1245743 2070 -15.T.G;74.T.- 0.792 0.347 6469515 2071 16.-.C;88.-.T 0.792 0.645 15241028 2072 -29.A.G;2.A.-;78.A.- 0.792 0.398 2711056 2073 0.T.-;2.A.C;82.A.G 0.791 0.747 1974296 2074 0.T.C;74.T.- 0.790 0.533 8637058 2075 66.CT.-G;86.-.G 0.789 0.254 8526611 2076 76.-.T;132.G.T 0.788 0.323 8144153 2077 76.G-;119.C.T 0.788 0.240 10566620 2078 15.-.T;73.A.C 0.788 0.613 8557775 2079 74.-.T;119.C.A 0.788 0.230 8462867 2080 79.GA.-T 0.787 0.613 8549438 2081 75.C.- 0.787 0.425 8558414 2082 74-.T;129.C.A 0.787 0.255 8105581 2083 76.GG-A;129.C.A 0.787 0.259 2281703 2084 0.T.-;86.C.T 0.786 0.719 2400499 2085 1.-.A;76.G.-;78.A.C 0.785 0.482 14920368 2086 -29.A.C;2.A.-;87.-.G 0.785 0.602 8543253 2087 75.-.G;91.A.-;93.A.G 0.785 0.452 8488707 2088 76.-.G;116.T.G 0.785 0.283 9979217 2089 19.-.G;86.-.C 0.783 0.612 15162226 2090 -29.A.G;86.-.A 0.783 0.522 12146137 2091 2.A.-;116.T.G 0.783 0.429 5454231 2092 8.G.C;76.G.- 0.782 0.646 2288382 2093 0.T.-;77.GA.--;83.A.T 0.781 0.648 8549424 2094 75.C.-;132.G.C 0.781 0.386 6461529 2095 16.-.C;85.T.- 0.781 0.720 1090544 2096 2.A.- 0.781 0.530 2282648 2097 0.T.-;84.-.T 0.779 0.667 12149194 2098 2.A.-;131.A.G 0.779 0.440 8142223 2099 76.G.-;124.T.G 0.779 0.273 8190575 2100 86.CC.-A 0.779 0.611 13854291 2281 -14.A.C;75.CG.-T 0.779 0.362 8092813 2282 75.-.A;121.C.A 0.778 0.281 8605540 2283 73.A.-;87.-.G 0.778 0.303 68946 2284 0.T.-;2.A.C 0.778 0.250 12199248 2285 2.A.-;76.GG.-T;132.G.C 0.778 0.424 8093073 2286 126.C.A;75.-.A 0.778 0.370 12149170 2287 2.A.-;131.A.C 0.776 0.577 447600 2288 -27.C.A;75.CG.-T 0.776 0.266 8143156 2289 76.G.-;126.C.T 0.776 0.346 1982252 2290 0.T.C;73.A.- 0.776 0.441 4255522 2291 4.T.-;115.T.G 0.776 0.764 8112417 2292 76.-.A;131.A.C 0.776 0.677 8083653 2293 74.-.G;121.C.A 0.775 0.434 8539008 2294 75.-.G;120.C.T 0.775 0.361 13750813 2295 -13.G.T;75.-.G 0.774 0.496 8759144 2296 55.-T;76.GG.-T 0.772 0.578 2684637 2297 0.T.-;2.A.C;131.AG.CC 0.771 0.251 8032414 2298 72.-.C 0.771 0.299 15165408 2299 -29.A.G;86.-.G 0.770 0.132 8352728 2300 86.C.-;129.C.A 0.770 0.200 12191702 2301 2.A.-;78.A.-;131.A.C 0.769 0.497 12751144 2302 0.-.T;74.-.T 0.769 0.417 2894079 2303 1.-.C;87.-.G 0.768 0.697 8480622 2304 78.A.-;129.C.A 0.768 0.332 8758901 2305 55.-.T;76.-.G 0.766 0.642 8202090 2306 87.-.A;121.C.A 0.766 0.622 2885067 2307 1.-.C;79.G.- 0.766 0.512 8202431 2308 87.-.A;132.G.C 0.765 0.537 12191659 2309 2.A.-;78.A.-;132.G.C 0.765 0.596 12149115 2310 2.A.-;133.A.C 0.764 0.439 2271200 2311 0.T.-;133.A.C 0.764 0.429 2252404 2312 0.T.-;74.T.G 0.763 0.476 8142993 2313 131.A.G;76.G.- 0.762 0.250 446438 2314 -27.C.A;78.A.- 0.762 0.249 8480581 2315 78.A.-;128.T.G 0.762 0.280 3133382 2316 1.T.G;3.C.-;74.-.G 0.761 0.629 2302762 2317 0.T.-;73.A.G 0.761 0.618 1041081 2318 -17.C.A;74.T.- 0.760 0.230 1074428 2319 -17.C.A;2.A.- 0.760 0.561 10571409 2320 15.-.T;65.GC.-T 0.760 0.639 8598575 2321 70.-.T;86.C.- 0.758 0.375 8363306 2322 87.-.T;131.A.C 0.757 0.452 8143881 2323 76.G.-;120.C.T 0.757 0.313 15159530 2324 -29.A.G;74.-.G 0.757 0.394 4230077 2325 4.T.-;75.C.A 0.756 0.733 8146649 2326 76.G.-;99.-.G 0.755 0.379 2684498 2327 0.T.-;2.A;130.T.G 0.755 0.295 8128273 2328 75.-.C;126.C.A 0.754 0.277 8066406 2329 74.T.-;126.C.A 0.752 0.237 8363243 2330 87.-.T;132.G.C 0.751 0.469 8142864 2331 76.G.-;132.GA.CC 0.751 0.276 2512825 2332 1.T.C;76.G.- 0.750 0.486 8091801 2333 75.-.A;115.T.G 0.750 0.260 1114939 2334 -16.C.A;76.G.- 0.749 0.264 8142311 2335 76.G.-;125.T.C 0.749 0.291 11774438 2336 2.-.C;76.GG.-A 0.748 0.658 15064284 2337 -29.A.G;1.TA.-- 0.748 0.383 1187746 2338 -15.T.G;0.T.- 0.748 0.384 8092581 2339 75.-.A;119.C.A 0.747 0.330 1246493 2340 -15.T.G;76.-.A 0.747 0.493 14646216 2341 -29.A.C;0.T.-;2.A.C;87.-.G 0.747 0.369 8142526 2342 76.G.-;127.T.C 0.746 0.249 8191621 2343 85.TCC.-GA 0.746 0.479 10308897 2344 17.-.T;78.A.G 0.745 0.691 14661314 2345 -29.A.C;0.T.-;2.A.G;75.-.C 0.745 0.570 8549337 2346 75.C.-;129.C.A 0.745 0.299 8753061 2347 55.-.T;79.G.- 0.745 0.514 10097262 2348 19.-.T;55.-.T 0.745 0.583 8161158 2349 79.G.-;131.A.C 0.744 0.215 2661991 2350 0.T.-;2.A.C;76.G.-;131.A.C 0.743 0.432 9987131 2351 19.-.G;86.C.- 0.743 0.684 1046156 2352 -17.C.A;76.CG.-T 0.743 0.206 3311900 2353 0.T.-;2.A.G;83.-.C 0.743 0.541 2412608 2354 1.-.A;76.GG.-T 0.742 0.454 8092717 2355 75.-.A;120.C.A 0.740 0.353 2684366 2356 0.T.-;2.A.C;128.T.G 0.740 0.320 8536239 2357 75.-.G;116.T.G 0.740 0.409 8483990 2358 78.A.-;98.-.T 0.739 0.635 1290147 2359 -15.T.G;2.A.-;76.G.- 0.737 0.358 8629656 2360 66.CT.-G;89.-.A 0.737 0.644 8039677 2361 72.-.G;86.-.C 0.736 0.628 8528174 2362 76.-.T;87.-.G 0.736 0.316 8142772 2363 76.G.-;130.T.C 0.736 0.350 12148593 2364 2.A.-;126.C.A 0.736 0.541 8089812 2365 75.-.A;88.G- 0.736 0.622 8436907 2366 81.GA.-T;131.A.C 0.734 0.289 6303279 2367 16.-.A;74.-.G 0.733 0.706 8136856 2368 76.G.-;88.G.- 0.732 0.393 13099840 2369 -1.GT.--;87.-.G 0.732 0.205 12147390 2370 2.A.-;119.C.A 0.731 0.364 8480707 2371 78.A.-;130.T.G 0.731 0.307 8145151 2372 76.G.-;113.A.C 0.729 0.240 2682115 2373 116.T.G;2.A.C;0.T.- 0.726 0.269 2397740 2374 1.-.A;73.-.A 0.775 0.570 8477975 2375 78.A.-;115.T.G 0.725 0.258 10190335 2376 18.-.G;99.-.G 0.725 0.472 15456232 2377 -30.C.G;76.GG.-T 0.725 0.153 1191613 2378 -15.T.G;0.T.-;2.A.C;76.G.- 0.724 0.396 8352265 2379 86.C.-;121.C.A 0.723 0.142 8212804 2380 86.-.C;130.T.G 0.722 0.481 8549476 2381 132.G.T;75.C.- 0.721 0.390 9994620 2382 19.-.G;77.-.T 0.721 0.613 14350752 2383 -25.A.C;76.GG-T 0.721 0.132 13099030 2384 -1.GT.-- 0.721 0.376 12147928 2385 2.A.-;121.C.A 0.721 0.488 1253117 2386 -15.T.G;74.-.T 0.720 0.253 8208073 2387 88.G.-;131.A.C 0.719 0.210 2684254 2388 0.T.-;2.A.C;127.T.G 0.719 0.353 8154688 2389 76.G.-;78.A.C;132.G.C 0.719 0.383 318717 2390 -28.G.C;76.G.- 0.719 0.192 8142885 2391 130.--T.TAG;133.A.G;76.G.- 0.719 0.301 14687527 2392 -29.A.C;4.T.-;78.A.- 0.718 0.527 15162677 2393 -29.A.G;89.-.A 0.718 0.668 15450951 2394 -30.C.G;76.GG.-C 0.717 0.477 8405267 2395 82.AA.-- 0.716 0.292 8066712 2396 74.T.-;132.G.T 0.716 0.310 8112393 2397 76.-.A;133.A.C 0.715 0.480 8564706 2398 75.CG.-T;120.C.A 0.715 0.237 8538090 2399 75.-.G;130.T.C 0.715 0.386 14081174 2400 -20.A.C;76.G.- 0.714 0.177 8357562 2401 87.-.G;126.C.A 0.713 0.285 6476171 2402 16.-.C;78.A.G 0.713 0.677 12145038 2403 2.A.-;115.T.G 0.713 0.524 8636717 2404 66.CT.-G;88.-.T 0.712 0.372 8208060 2405 88.G.-;132.G.T 0.712 0.261 2746161 2406 0.T.-;2.C;66.CT.-G;132.G.C 0.711 0.362 8064859 2407 74.T.-;115.T.G 0.711 0.210 1981797 2408 0.T.C;75.CG.-T 0.711 0.646 15719823 2409 -32.G.T;0.T.-;2.A.C 0.710 0.271 3024059 2410 1.TA.--;82.AA.-C 0.710 0.373 14806152 2411 -29.A.C;89.-.C 0.709 0.182 14634677 2412 -29.A.C;0.T.-;76.G.- 0.708 0.421 672656 2413 -23.C.A;75.-.G 0.708 0.430 8628797 2414 66.CT.-G;77.GA.-- 0.708 0.333 10529623 2415 15.-.T;85.TC.-A 0.708 0.506 10196969 2416 18.-.G;78.A.- 0.707 0.698 8057272 2417 73.-.A;121.C.A 0.707 0.370 13845728 2418 -14.A.C;75.-.C 0.707 0.297 1045822 2419 -17.C.A;76.-.G 0.706 0.324 10460865 2420 16.C.-;76.GG.-C 0.706 0.523 4222138 2421 4.T.-72.-.G 0.705 0.401 1152457 2422 -15.T.C;0.T.-;2.A.C 0.704 0.351 8069945 2423 74.T.-;87.-.T 0.704 0.402 6303440 2424 16.-.A;75.-.A 0.704 0.657 5593794 2425 10.T.C;75.CG.-T 0.704 0.281 14654654 2426 -29.A.C;1.TA.-- 0.703 0.363 7829345 2427 55.-.G;76.GG.-C 0.703 0.651 7490581 2428 36.C.A;76.GG.-C 0.703 0.439 15452184 2429 -30.C.G;86.-.C 0.702 0.465 8089736 2430 75.-.A;87.-.A 0.702 0.404 3161365 2431 0.T.-;2.A.G;14.-.A 0.702 0.700 8215458 2432 88.GA.-C 0.702 0.286 2455947 2433 1.TA.--;3.C.A;73.-.A 0.702 0.693 827787 2434 -21.C.A;76.G.- 0.702 0.246 3574182 2435 2.-.A;55.-.G 0.701 0.681 8504697 2436 78.-.T 0.701 0.457 8147538 2437 76.G.-;91.A.-;93.A.G 0.701 0.391 8436856 2438 81.GA.-T;132.G.C 0.700 0.199 8110287 2439 76.-.A;86.-.C 0.700 0.448 8598693 2440 70.-.T;87.-.T 0.700 0.315 4260194 2441 4.T.-;129.C.T 0.699 0.510 8059622 2442 73.-.A;87.-.G 0.699 0.389 8586230 2443 73.AT.-G 0.699 0.265 8126524 2444 75.-.C;115.T.G 0.699 0.336 10084621 2445 19.-.T;82.AA.-T 0.699 0.642 10607021 2446 16.C.T;78.A.- 0.698 0.567 8212230 2447 86.-.C;120.C.A 0.698 0.505 2664493 2448 0.T.-;2.A.C;79.G.A 0.698 0.640 2203429 2449 0.T.-;18.C.- 0.698 0.407 8605503 2450 73.A.-;86.C.- 0.697 0.200 13852662 2451 -14.A.C;78.A.- 0.697 0.309 8546163 2452 75.C.-;86.-.C 0.697 0.445 446575 2453 -27.C.A;76.-.G 0.696 0.351 8065997 2454 74.T.-;120.C.A 0.696 0.234 11888602 2455 2.A.C;75.-.G 0.696 0.515 8536608 2456 75.-.G;118.T.C 0.694 0.323 14797194 2457 -29.A.C;74.-.G 0.694 0.384 15166776 2458 -29.A.G;82.AA.-T 0.694 0.237 14800643 2459 -29.A.C;77.GA.-- 0.693 0.379 8030604 2460 72.-.C;86.-.C 0.692 0.345 2464748 2461 1.TA.--;3.C.A;82.AA.-C 0.692 0.574 8493269 2462 76.-.G;99.-.G 0.691 0.356 8549456 2463 75.C.-;133.A.C 0.691 0.458 2307776 2464 0.T.-;66.CT.-- 0.690 0.673 6306305 2465 16.-.A;86.-.C 0.690 0.602 8126956 2466 75.-.C;116.T.G 0.690 0.278 14809754 2467 -29.A.C;81.GA.-T 0.688 0.296 8212714 2468 86.-.C;128.T.G 0.688 0.369 1251890 2469 -15.T.G;78.A.- 0.687 0.319 8518607 2470 76.GG.-T;119.C.A 0.687 0.191 8057702 2471 73.-.A;131.A.C 0.686 0.432 3024866 2472 1.TA.--;82.AA.-G 0.686 0.454 8367599 2473 86.-.G;133.A.C 0.686 0.157 8431922 2474 82.AA.-T 0.686 0.217 8144351 2475 76.G.-;117.G.T 0.685 0.239 8538257 2476 75.-.G;131.A.C;133.A.C 0.685 0.419 8543064 2477 75.-.G;91.A.- 0.685 0.640 15455856 2478 -30.C.G;76.-.G 0.685 0.299 12149015 2479 2.A.-;130.T.G 0.685 0.459 2685087 2480 0.T.-;2.A.C;122.A.C 0.684 0.234 8084140 2481 74.-.G;132.G.C 0.683 0.396 8142757 2482 76.G.-;130.T.C;132.G.C 0.683 0.272 8538197 2483 75.-.G;134.G.T 0.683 0.368 15058053 2484 -29.A.G;0.T.-;2.A.C;76.GG.-C 0.683 0.336 8066567 2485 74.T.-;129.C.A 0.681 0.266 441402 2486 -27.C.A;74.T.- 0.681 0.300 1042785 2487 -17.C.A;86.-.C 0.679 0.335 8490149 2488 76.-.G;127.T.G 0.678 0.293 1905560 2489 0.TTA.---;3.C.A;87.-.A 0.678 0.635 8352170 2490 86.C.-;120.C.A 0.678 0.182 1252598 2491 -15.T.G;76.-.T 0.678 0.235 2400384 2492 1.-.A;77.-.A 0.678 0.356 8087722 2493 74.-.G;86.C.- 0.676 0.432 8101522 2494 75.-C.AG 0.676 0.285 8087834 2495 74.-.G;87.-.T 0.676 0.449 8431908 2496 82.AA.-T;132.G.C 0.676 0.225 14645411 2497 -29.A.C;0.T.-;2.A.C;86.-.C 0.676 0.635 2835829 2498 0.T.-;2.A.C;6.G.T 0.675 0.298 8438736 2499 81.GAA.-TC 0.674 0.360 8065838 2500 74.T.-;119.C.A 0.673 0.209 15171004 2501 -29.A.G;73.A.- 0.673 0.259 8084203 2502 74.-.G131.A.C 0.673 0.327 15161712 2503 -29.A.G;77.GA.-- 0.672 0.388 6613064 2504 18.C.-;77.-.A 0.672 0.551 12315000 2505 2.A.-;15.-.T;75.-.G 0.672 0.635 14246167 2506 -24.G.T;75.-.G 0.672 0.308 15051656 2507 -29.A.G;0.T.- 0.671 0.366 8469914 2508 78.-.C;121.C.A 0.671 0.232 8352836 2509 86.C.-;133.A.C 0.670 0.207 8554990 2510 74.-.T;87.-.A 0.670 0.490 830076 2511 -21.C.A;75.-.G 0.670 0.422 8538376 2512 75.-.G;126.C.G 0.670 0.370 15451096 2513 -30.C.G;75.-.C 0.670 0.236 1290476 2514 -15.T.G;2.A.- 0.669 0.658 14644913 2515 -29.A.C;0.T.-;2.A.C;75.-.C 0.668 0.335 8481064 2516 78.A.-;123.A.C 0.667 0.232 12726534 2517 0.-.T;86.-.C 0.666 0.531 14814019 2518 -29.A.C;75.C.- 0.666 0.397 15450607 2519 -30.C.G;75.-.A 0.665 0.225 8512477 2520 76.G.-;78.A.T;132.G.C 0.665 0.478 1247921 2521 -15.T.G;87.-.A 0.665 0.476 6461965 2522 16.-.C;86.CC.-A 0.664 0.620 14815751 2523 -29.A.C;73.A.G 0.663 0.362 8557906 2524 74.-.T;120.C.A 0.663 0.196 8174025 2525 77.GA.--;132.G.T 0.663 0.265 1979872 2526 0.T.C;78.-.C 0.66;3 0.404 8148116 2527 76.G.-;87.-.T 0.662 0.584 8055441 2528 73.-.A;86.-.C 0.662 0.471 15162449 2529 -29.A.G;88.G.- 0.662 0.206 8522485 2530 76.GGA.-TC 0.662 0.401 3081068 2531 1.TA.--;18.-.G 0.662 0.556 8117952 2532 76.GG.-C;126.C.A 0.661 0.381 6469397 2533 16.-.C;89.-.T 0.661 0.591 8181855 2534 85.TCC.-AA 0.661 0.568 1044315 2535 -17.C.A;86.C.- 0.661 0.167 14920528 2536 -29.A.C.;2.A.-;82.A.- 0.659 0.536 8518772 2537 76.GG-T;120.C.A 0.659 0.283 15058093 2538 -29.A.G;0.T.-;2.A.C;75.-.C 0.658 0.434 8057683 2539 132.G.T;73.-.A 0.657 0.434 2459622 2540 1.TA.--;3.C.A;86.-.A 0.656 0.656 8069836 2541 74.T.-;86.C.- 0.656 0.293 3320802 2542 2.A.G;0.T.-;80.A.- 0.656 0.611 14919186 2543 -29.A.C;2.A.-;77.GA.-- 0.655 0.360 8207846 2544 88.G.-;126.C.A 0.655 0.244 447068 2545 -27.C.A;76.-.T 0.655 0.227 8603132 2546 73.A.-;132.G.C 0.654 0.247 8755264 2547 55.-.T;132.G.C 0.654 0.548 443309 2548 -27.C.A;86.-.C 0.653 0.447 8548846 2549 75.C.-;121.C.A 0.653 0.455 815029 2550 77.-.A;132.G.T 0.652 0.274 8603165 2551 73.A.-;133.A.C 0.652 0.298 12312790 2552 16.C.-;2.A.- 0.652 0.524 10248608 2553 18.C.T;76.G.- 0.651 0.536 1046713 2554 -17.C.A;75.CG.-T 0.651 0.263 8638044 2555 66.CT.-G;82.AA.-T 0.651 0.287 3315325 2556 0.T.-;2.A.G;82.AA.-C 0.650 0.605 12314014 2557 2.A.-;15.-.T;76.G.- 0.649 0.574 8494400 2558 76.-.G;86.C.- 0.649 0.187 14920881 2559 -29.A.C;2.A.-;80.A.- 0.648 0.517 14243707 2560 -24.G.T;76.G.- 0.648 0.185 12148911 2561 2.A.-;129.C.A 0.647 0.601 12149062 2562 2.A.-;132.G.C 0.646 0.502 8600526 2563 73.A.-;88.G- 0.645 0.440 8538871 2564 75.-.G;121.C.T 0.645 0.402 8603181 2565 73.A.-;132.G.T 0.645 0.289 15450764 2566 -30.C.G;76.GG.-A 0.644 0.211 12149230 2567 2.A.-;129.C.G 0.643 0.340 8558338 2568 74.-.T;127.T.G 0.643 0.272 8367575 2569 86.-.G;132.G.C 0.642 0.146 14647726 2570 -29.A.C;0.T.-;2.A.C;66.CT.-G 0.641 0.378 8490463 2571 76.-.G;131.AG.CC 0.640 0.222 12123507 2572 2.A.-;76.G.-;121.C.A 0.640 0.452 8352850 2573 86.C.-;132.G.T 0.640 0.245 12191691 2574 2.A.-;78.A.-;132.G.T 0.639 0.499 8638264 2575 66.CT.-G;80.A.- 0.639 0.282 1195928 2576 -15.T.G;1.TA.-- 0.639 0.361 1979286 2577 0.T.C;81.GA.-T 0.639 0.548 8207662 2578 88.G.-;121.C.A 0.638 0.120 6460643 2579 16.-.C;81.G- 0.638 0.572 2686745 2580 0.T.-;2.A.C;113.A.C 0.638 0.276 1045705 2581 -17.C.A;78.A.- 0.638 0.262 8600457 2582 73.A.-;87.-.A 0.636 0.454 7948057 2583 66.CT.-A;76.-.G 0.636 0.380 10091271 2584 19.-T;73.AT.-C 0.636 0.542 442030 2585 -27.C.A;76.-.A 0.636 0.592 844891 2586 2.A.-;-21.C.A 0.633 0.622 10516019 2587 15.-.T;71.-.C 0.633 0.534 12016332 2588 2.A.-;18.C.- 0.632 0.463 8073253 2589 74.-.C;132.G.C; 0.632 0.356 8357699 2590 87.-.G;128.T.G 0.630 0.335 2684905 2591 0.T.-;2.A.C;123.A.C 0.630 0.301 2684593 2592 0.T.-;2.A.C;134.G.T 0.630 0.258 12149142 2593 2.A.-;132.G.T 0.630 0.481 2881692 2594 1.-.C;74.-.C 0.628 0.531 5590003 2595 87.-.G;10.T.C 0.628 0.471 12123808 2596 132.G.T;2.A.-;76.G.- 0.628 0.327 8212595 2597 86.-.C;126.C.A 0.627 0.514 8173470 2598 77.GA.--;121.C.A 0.627 0.292 8034488 2599 72.-.C;82.A.- 0.627 0.141 2411142 2600 1.-.A;78.-.C 0.626 0.400 8096384 2601 75.-.A;82.A.- 0.626 0.418 2723173 2602 0.T.-;2.A.C;76.-.G;132.G.C 0.626 0.320 8118097 2603 76.GG.-C;128.T.G 0.625 0.405 8543409 2604 75.-.G;91.AA.-G 0.625 0.400 14812614 2605 -29.A.C;76.G.-;78.A.T 0.625 0.410 6476723 2606 16.-.C;76.G.-;78.A.T 0.624 0.568 8519286 2607 76.GG.-T;127.T.G 0.624 0.239 8501650 2608 78.AG.-T 0.623 0.440 8208050 2609 88.G.-;133.A.C 0.623 0.206 8549499 2610 75.C.-;131.A.C 0.623 0.381 12009703 2611 2.A.-;17.-.A 0.673 0.617 8128850 2612 75.-.C;123.A.C 0.623 0.272 1862825 2613 0.TT.--;78.-.T 0.622 0.588 6368672 2614 17.-.A;78.-.C 0.622 0.607 8519348 2615 76.GG.-T;128.T.G 0.622 0.277 1041692 2616 -17.C.A;76.GG.-C 0.622 0.482 8018631 2617 72.-.A 0.621 0.469 8066533 2618 74.T.-;128.T.G 0.619 0.261 8436892 2619 81.GA.-T;132.G.T 0.619 0.154 8636610 2620 66.CT.-G;89.A.- 0.618 0.524 2884910 2621 1.-.C;77.-.C 0.617 0.494 8143053 2622 76.G.-;129.C.T 0.617 0.285 8356385 2623 87.-.G;115.T.G 0.616 0.348 8561418 2624 74.-.T;87.-.T 0.616 0.531 6467416 2625 16.-.C;99.-.G 0.615 0.507 2723199 2626 0.T.-;2.A.C;76.-.G;132.G.T 0.615 0.389 13746674 2627 -13.G.T;75.-.C 0.614 0.317 15736191 2628 -32.G.T;76.G.- 0.614 0.181 2950619 2629 1.TA.--;17.T.C 0.613 0.330 1250048 2630 -15.T.G;87.-.G 0.612 0.301 8519441 2631 76.GG.-T;130.T.G 0.611 0.227 8174044 2632 77.GA.--;131.A.C 0.611 0.368 8083913 2633 74.-.G;126.C.A 0.610 0.361 6554290 2634 18.C.A;75.-.C 0.610 0.248 8481228 2635 78.A.-;122.A.C 0.610 0.293 14004700 2636 -19.G.T;0.T.-;2.A.C 0.610 0.268 481605 2637 -27.C.A;2.A.- 0.610 0.487 2262447 2638 0.T.-;81.GA.-C 0.608 0.518 2683891 2639 0.T.-;2.A.C;124.T.G 0.608 0.300 2685505 2640 0.T.-;2.A.C;120.C.T 0.608 0.287 827692 2641 -21.C.A;75.-.C 0.608 0.315 13101663 2642 -1.GT.--;74.-.T 0.607 0.272 2271017 2643 0.T.-;128.T.G 0.607 0.345 8066699 2644 74.T.-;133.A.C 0.607 0.229 8118193 2645 76.GG.-C;130.T.G 0.607 0.534 8073290 2646 74.-.C;132.G.T 0.606 0.307 1117646 2647 -16.C.A;75.-.G 0.606 0.417 444910 2648 -27.C.A;86.C.- 0.605 0.107 8563682 2649 75.CG.-T;115.T.G 0.605 0.210 14645196 2650 -29.A.C;0.T.-;2.A.C;77.GA.-- 0.604 0.451 14663089 2651 -29.A.C;0.T.-;2.A.G;76.-.G 0.604 0.579 8480843 2652 78.A.-;131.A.C;133.A.C 0.603 0.221 15241063 2653 -29.A.G;2.A.-;76.-.G 0.603 0.535 8128359 2654 75.-.C;127.T.G 0.603 0.246 12202830 2655 2.A.-;75.-.G;131.A.C 0.602 0.300 2516661 2656 1.T.C;76.-.G 0.602 0.569 8600854 2657 73.A.-;98.-.A 0.601 0.555 15158807 2658 -29.A.G;73.-.A 0.600 0.594 12147720 2659 2.A.-;120.C.A 0.600 0.524 14344554 2660 -25.A.C;76.GG.-A 0.600 0.212 3133295 2661 1.T.G;3.C.-;74.T.- 0.600 0.541 3601058 2662 2.-.A;76.GG.-T 0.599 0.520 8562045 2663 74.-.T;82.AA.-T 0.599 0.257 8080686 2664 74.-.G89.-.A 0.599 0.542 8116266 2665 76.GG.-C;115.T.G 0.599 0.439 8528148 2666 76.-.T;86.C.- 0.598 0.268 14809572 2667 -29.A.C;82.AA.-T 0.597 0.169 1041548 2668 -17.C.A;76.GG.-A 0.597 0.348 13847372 2669 -14.A.C;86.-.C 0.597 0.440 2654872 2670 0.T.-;2.A.C;75.C.A 0.596 0.361 8543705 2671 75.-.G;89.A.G 0.596 0.481 8150315 2672 77.-.A;131.A.C 0.595 0.217 13854171 2673 -14.A.C;74.-.T 0.595 0.255 8084187 2674 74.-.G;132.G.T 0.595 0.378 1249988 2675 -15.T.G;86.C.- 0.594 0.264 10308807 2676 17.-.T;78.A.-;80.A.- 0.593 0.538 8093276 2677 75.-.A;130.T.G 0.593 0.294 15069677 2678 -29.A.G;0.T.-;2.A.G;75.-.G 0.593 0.429 2884699 2679 1.-.C;77.-.A 0.593 0.444 14921605 2680 -29.A.C;2.A.-;74.-.T 0.592 0.536 8448153 2681 80.A.-;132.G.C 0.592 0.175 8140966 2682 76.G.-;118.T.C 0.591 0.209 8161100 2683 79.G.-;132.G.C 0.591 0.221 15165008 2684 -29.A.G;88.-.T 0.590 0.294 15058006 2685 -29.A.G;0.T.-;2.A.C;76.GG.-A 0.590 0.449 14647360 2686 -29.A.C;0.T.-;2.A.C;75.CG.-T 0.589 0.365 8207961 2687 88.G.-;129.C.A 0.588 0.254 2684707 2688 0.T.-;2.A.C;129.C.G 0.587 0.249 12177699 2689 2.A.-82.A.-;84.A.T 0.587 0.578 8495115 2690 76.-.G;80.A.G 0.587 0.277 8173741 2691 77.GA.--;126.C.A 0.586 0.262 8044380 2692 72.-.G;87.-.G 0.586 0.496 2270366 2693 0.T.-;120.C.A. 0.585 0.348 15456767 2694 -30.C.G;74.-.T 0.585 0.259 12752882 2695 0.-.T;73.AT.-G 0.584 0.561 4217308 2696 4.T.-;71.T.C 0.584 0.515 14810890 2697 -29.A.C;78.AG.-C 0.583 0.368 13853442 2698 -14.A.C;76.GG.-T 0.583 0.211 8448176 2699 80.A.- 0.583 0.209 8103057 2700 76.GG.-A;98.-.A 0.582 0.554 8141130 2701 76.G.-;118.T.G 0.581 0.262 8133120 2702 75.-.C;86.-.G 0.581 0.269 14921140 2703 -29.A.C;2.A.-;76.-.G 0.581 0.464 1046627 2704 -17.C.A;74.-.T 0.581 0.238 8490817 2705 76.-.G;122.A.C 0.581 0.338 2749021 2706 0.T.-;2.A.C;65.G.T 0.581 0.520 1251730 2707 -15.T.G;78.-.C 0.580 0.278 8565400 2708 75.CG.-T;131.AG.CC 0.580 0.163 8034315 2709 72.-.C;87.-.G 0.580 0.400 1095467 2710 -16.C.A;0.T.-;2.A.C 0.578 0.254 1982142 2711 0.T.C;70.-.T 0.578 0.515 2661968 2712 0.T.-;2.A.C;76.G.-;133.A.C 0.577 0.442 14529775 2713 -28.G.T;75.-.G 0.577 0.358 2464540 2714 0.T.-;3.C.-;82.AA.-- 0.576 0.497 3011533 2715 1.TA.--;126.C.A 0.576 0.386 8160673 2716 79.G.-;121.C.A 0.576 0.277 445036 2717 -27.C.A;87.-.T 0.576 0.386 8480668 2718 78.A.-;130.T.C 0.576 0.239 446329 2719 -27.C.A;78.-.C 0.576 0.276 8524684 2720 76.-.T;86.-.C 0.575 0.428 14350148 2721 -25.A.C;78.A.- 0.575 0.252 15456629 2722 -30.C.G;75.C.- 0.575 0.433 8084175 2723 74.-.G;133.A.C 0.574 0.498 8470281 2724 78.-.C;133.A.C 0.574 0.327 1976159 2725 0.T.C;88.G.- 0.573 0.487 2553815 2726 0.T.-;2.A.C;11.T.C 0.573 0.381 856531;3 2727 75.CG.-T;130.T.G 0.573 0.285 8142626 2728 76.G.-128.T.C 0.573 0.271 15059444 2729 -29.A.G;0.T.-;2.A.C;76.GG.-T 0.571 0.539 14349990 2730 -25.A.C;78.-.C 0.570 0.340 7944404 2731 66.CT.-A;86.-.C 0.570 0.517 8143508 2732 76.G.-;122.A.G 0.570 0.295 8483736 2733 78.A.-;99.-.G 0.570 0.383 8457128 2734 80.AG.-T 0.570 0.408 14685680 2735 -29.A.C;4.T.-;76.GG.-C 0.570 0.468 8639135 2736 66.CT.-G;75.-.G 0.570 0.439 8093196 2737 75.-.A;128.T.G 0.570 0.286 2574670 2738 0.T.-;2.A.C;21.T.A 0.569 0.278 2270511 2739 0.T.-;121.C.A 0.569 0.347 2411434 2740 1.-.A;78.A.- 0.568 0.492 8128649 2741 75.-.C;131.A.C;133.A.C 0.568 0.311 2837903 2742 2.A.C;0.T.-;5.G.T 0.567 0.302 15456872 2743 -30.C.G;75.CG.-T 0.567 0.275 2684575 2744 130.-- 0.567 0.297 T.TAG;133.A.G;2.A.C;0.T.- 15486653 2745 -30.C.G;2.A.- 0.567 0.457 12202811 2746 2.A.-;75.-.G;133.A.C 0.566 0.396 8480879 2747 78.A.-;129.C.G 0.566 0.324 3011188 2748 1.TA.--;121.C.A 0.564 0.372 8297879 2749 99.-.G 0.563 0.268 8352639 2750 86.C.-;127.T.G 0.563 0.202 14801514 2751 -29.A.C;86.-.A 0.562 0.474 1975537 2752 0.T.C;79.G.- 0.562 0.486 8480783 2753 78.A.-;134.G.T 0.561 0.409 14351204 2754 -25.A.C;75.C.- 0.561 0.404 1042672 2755 -17.C.A;87.-.A 0.560 0.387 8480385 2756 78.A.-;126.C.A 0.560 0.238 8105496 2757 76.GG.-A;127.T.G 0.559 0.269 15059173 2758 -29.A.G;0.T.-;2.A.C;80.A.- 0.558 0.364 8132470 2759 75.-.C;91.AA.-G 0.558 0.468 14663399 2760 -29.A.C;0;T.-;2.A.G;75.C.- 0.556 0.453 8132353 2761 75.-.C;91.A.-;93.A.G 0.556 0.392 6557204 2762 18.C.A;78.A- 0.555 0.330 13845080 2763 -14.A.C;75.-.A 0.554 0.281 2894429 2764 1.-.C;86.-.G 0.554 0.356 8605594 2765 73.A.-;87.-.T 0.553 0.323 14918668 2766 -29.C;2.A.-;75.-.A 0.553 0.285 13852859 2767 -14.A.C;76.-.G 0.553 0.304 8558273 2768 74.-.T;126.C.A 0.553 0.203 14344734 2769 -25.A.C;76.GG.-C 0.552 0.425 8063226 2770 74.T.-;87.-.A 0.552 0.355 8564564 2771 75.CG.-T;119.C.A 0.552 0.230 13687669 2772 -12.G.T;75.-.G 0.551 0.378 14812439 2773 -29.A.C;78.A.T 0.551 0.502 7944045 2774 66.CT.-A;76.G.- 0.551 0.426 2685752 2775 0.T.-;2.A.C;119.C.T 0.549 0.206 8118242 2776 130.-- 0.549 0.423 T.TAG;133.A.G;76.GG.-C 1245577 2777 -15.T.G;73.-.A 0.549 0.539 15454032 2778 -30.C.G;86.C.- 0.548 0.147 15738375 2779 -32.G.T;75.-.G 0.548 0.300 6302341 2780 16.-.A;72.-.C 0.548 0.363 2287278 2781 0.T.-;82.-.T 0.548 0.435 3599083 2782 2.-.A;78.-.C 0.548 0.398 8538303 2783 75.-.G;129.C.G 0.547 0.446 3025181 2784 1.TA.--;82.-.T 0.546 0.498 999582 2785 -17.C.A;0.T.- 0.546 0.407 9986114 2786 19.-.G;89.-.C 0.546 0.492 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75.-.G;132.G.A 0.467 0.349 8519808 2955 76.GG.-T;122.A.C 0.467 0.179 8538739 2956 75.-.G;122.A.G 0.467 0.335 8055399 2957 73.-.A;88.G.- 0.466 0.320 8602922 2958 73.A.-;126.C.A 0.466 0.283 8558390 2959 74.-.T;128.T.G 9.465 0.206 8202371 2960 87.-.A;129.C.A 0.465 0.465 8495023 2961 78.A.-;82.A.G 0.463 0.212 8093252 2962 75.-.A;130.T.C 0.463 0.335 2566367 2963 0.T.-;2.A.C;17.T.C 0.461 0.268 443194 2964 -27.C.A;87.-.A 0.461 0.399 8586216 2965 73.AT.-G;132.G.C 0.461 0.251 8492129 2966 76.-.G;113.A.G 0.460 0.274 8602593 2967 73.A.-120.C.A 0.460 0.167 12438314 2968 1.TAC.---;76.-.T 9.459 0.409 8018666 2969 72.-.A;131.A.C 0.459 0.406 2658141 2970 0.T.-;2.A.C;76.GG.- 0.459 0.418 C;132.G.C 2270855 2971 0.T.-;126.C.A 0.458 0.340 3011711 2972 1.TA.--;129.C.A 0.458 0.369 8357785 2973 87.-.G;130.T.G 0.457 0.321 12148855 2974 2.A.-;128.T.G 0.457 0.424 8538425 2975 75.-.G;126.C.T 0.456 0.392 14812176 2976 -29.A.C;78.AG.-T 0.455 0.422 959345 2977 -18.T.G;0.T.-;2.A.C 0.455 0.263 8352569 2978 86.C.-;126.C.A 0.452 0.232 8562579 2979 75.CG.-T;86.-.C 0.452 0.285 12185280 2980 2.A.-;80.A.-;132.G.C 0.452 0.397 8118567 2981 76.GG.-C;122.A.C 0.449 0.341 8129443 2982 75.-.C;119.C.T 0.448 0.241 8488242 2983 76.-.G;115.T.G 0.448 0.303 2685947 2984 0.T.-;2.A.C;117.G.T 0.447 0.224 2684042 2985 0.T.-;2.A.C;125.T.G 0.446 0.225 2628011 2986 0.T.-;2.A.C;65.G.A 0.446 0.431 1093922 2987 -16.C.A;0.T.- 0.446 0.385 14021392 2988 -19.G.T;76.G.- 0.445 0.211 14023783 2989 -19.G.T;75.-.G 0.445 0.321 8479108 2990 118.T.C;78.A.- 0.444 0.180 4295742 2991 4.T.-;78.A.-;132.G.T 0.444 0.342 8348822 2992 88.-.T;132.G.C 0.444 0.307 8448031 2993 80.A.-;128.T.G 0.443 0.216 8480854 2994 78.A.-131.A.G 0.442 0.339 8073282 2995 74.-.C;133.A.c 0.442 0.352 2271058 2996 129.C.A;0.T.- 0.442 0.317 12151722 2997 2.A.-;113.A.C 0.441 0.449 13168765 2998 -1.G.T;76.G.- 0.440 0.238 8760885 2999 56.G.T;76.G.- 0.439 0.164 8518019 3000 76.GG.-T;116.T.G 0.438 0.236 1117245 3001 -16.C.A;78.A.- 0.438 0.168 8592769 3002 70.-.T;88.G.- 0.438 0.245 8628663 3003 66.CT.-G;79.G.- 0.438 0.183 8480752 3004 78.A.-;132.GA.CC 0.438 0.249 8059585 3005 73.-.A;86.C.- 0.437 0.436 13750261 3006 -13.G.T;78.A.- 0.437 0.253 8539599 3007 75.-.G;114.G.T 0.437 0.374 8352028 3008 86.C.-;119.C.A 0.436 0.189 8129947 3009 75.-.C;113.A.C 0.436 0.305 8538081 3010 75.-.G;130.T.C;132.G.C 0.435 0.332 8561460 3011 74.-.T.86.-.G 0.433 0.233 8363222 3012 87.-.T;130.T.G 0.432 0.345 15749286 3013 -32.G.T;2.A.- 0.431 0.390 8129269 3014 75.-.C;120.C.T 0.431 0.274 445858 3015 -27.C.A;82.AA.-T 0.431 0.234 8133915 3016 75.-.C;80.A.G 0.431 0.344 1045161 3017 -17.C.A;82.AA.-T 0.430 0.182 2569551 3018 0.T.-;2.A.C;18.C.A 0.430 0.278 8034268 3019 72.-.C;86.C.- 0.428 0.226 481315 3020 -27.C.A;2.A.-;76.G.- 0.428 0.366 447361 3021 -27.C.A;75.C.- 0.427 0.372 393117 3022 -27.C.A;0.T.-;2.A.C;76.G.- 0.427 0.380 672550 3023 -23.C.A;76.GG.-T 0.427 0.135 13171223 3024 -1.G.T;78.A.- 0.427 0.170 2269114 3025 0.T.-;115.T.G 0.424 0.334 15164751 3026 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14647317 3073 -29.A.C;0.T.-;2.A.C;74.-.T 0.399 0.337 8431948 3074 82.AA.-T;132.G.T 0.396 0.283 14344384 3075 -25.A.C;75.-.A 0.396 0.313 8508448 3076 78.A.T;132.G.C 0.395 0.355 8150265 3077 77.-.A;132.G.C 0.395 0.232 8654330 3078 65.GC.-T;78.A.- 0.395 0.294 8093514 3079 75.-.A;123.A.C 0.394 0.309 8352775 3080 86.C.-;130.TG 0.392 0.217 8066628 3081 74.T.-;130.T.G 0.392 0.262 15168618 3082 -29.A.G;76.G.-;78.A.T 0.390 0.336 672344 3083 -23.C.A;78.A.- 0.390 0.322 8586257 3084 73.AT.-G;132.G.T 0.388 0.296 8105301 3085 76.GG.-A;124.T.G 0.388 0.288 8212901 3086 86.-.C;131.AG.CC 0.386 0.353 13588657 3087 -10.A.C;76.G.- 0.385 0.348 728974 3088 -22.T.A;75.-.G 0.384 0.325 8448212 3089 80.A.-;132.G.T 0.383 0.198 8128219 3090 75.-.C;125.T.G 0.382 0.342 8084164 3091 130.--T.TAG;133.A.G;74.-.G 0.381 0.324 13800992 3092 -14.A.C;1.TA.-- 0.381 0.380 8084111 3093 74.-.G;130.T.G 0.380 0.285 14348272 3094 -25.A.C;87.-.G 0.376 0.227 8032112 3095 72.-.C;121.C.A 0.375 0.317 8599500 3096 70.-.T;80.A.- 0.375 0.307 14647476 3097 -29.A.C;0.T.-;2.A.C;73.AT.-G 0.375 0.287 8637349 3098 66.CT-G;82.A.- 0.375 0.370 14059318 3099 2.A.C;0.T.-;-20.A.C 0.374 0.261 5590089 3100 10.T.C;87.-.T 0.373 0.345 8105685 3101 76.GG-A;130.-- 0.372 0.233 T.TAG;133.A.G 2687214 3102 0.T.-;2.A.C;113.A.G 0.371 0.260 8605752 3103 73.A.-;82.A.- 0.369 0.345 8066727 3104 74.T.-;131.AG.CC 0.367 0.285 872410 3105 -21.C.-;76.G- 0.366 0.282 13168637 3106 -1.G.T;75.-.C 0.366 0.326 442575 3107 -27.C.A;77.-.A 0.365 0.149 670080 3108 -23.C.A;76.GG.-A 0.365 0.229 2536818 3109 1.T.C;3.C.- 0.365 0.278 15239473 3110 -29.A.G;2.A.-;75.-.A 0.364 0.308 8599361 3111 70.-.T;82.AA.-T 0.364 0.203 8447558 3112 80.A.-;121.C.A 0.364 0.190 8032400 3113 72.-.C;132.G.C 0.363 0.277 2591751 3114 0.T.-;2.A.C;33.C.A 0.363 0.290 851955 3115 76.G.-;82.A.G 0.362 0.293 829720 3116 -21.C.A;78.A.- 0.362 0.340 8633205 3117 66.CT-G;133.A.C 0.361 0.178 8367621 3118 86.-.G;131.A.C 0.361 0.150 8652746 3119 65.GC.-T 0.360 0.341 8641968 3120 66.CT.-- 0.360 0.335 8489994 3121 76.-.G;125.T.G 0.359 0.243 2271196 3122 0.T.-;134.G.T 0.357 0.333 2684526 3123 0.T.-;2.A.C;132.G.A 0.357 0.211 6557839 3124 18.C.A;74.-.T 0.356 0.194 15057882 3125 -29.A.G;0.T.-;2.A.C;74.T.- 0.356 0.348 14812029 3126 -29.A.C;78.A.G 0.355 0.332 8565161 3127 75.CG.-T;127.T.G 0.354 0.290 1042365 3128 -17.C.A;77.GA.-- 0.352 0.264 1114842 3129 -16.C.A;75.-.C 0.351 0.323 3011677 3130 1.TA.--;128.T.G 0.349 0.272 8367521 3131 86.-.G;129.C.A 0.349 0.129 8545111 3132 75.-.G;82.A.G 0.349 0.279 13670603 3133 -12.G.T;0.T.-;2.A.C 0.347 0.221 8152309 3134 76.G-;80.A.G 0.345 0.240 14635704 3135 -29.C;0.T.-;78.A.- 0.344 0.269 8101708 3136 75.CGG.-AT 0.344 0.263 15738145 3137 -32.G.T;76.-.G 0.343 0.283 14351983 3138 -25.A.C;73.A.- 0.342 0.318 8066472 3139 74.T.-;127.T.G 0.341 0.219 8134358 3140 75.-G.CT 0.341 0.260 8603055 3141 73.A.-;129.C.A 0.340 0.285 1251152 3142 -15.T.G;82.AA.-T 0.337 0.222 1005071 3143 -17.C.A;1.TA.-- 0.335 0.306 8137618 3144 76.G.-;104.C.A 0.335 0.191 15158102 3145 -29.A.G;72.-.C 0.335 0.245 8129152 3146 75.-.C;121.C.T 0.334 0.186 8208002 3147 88.G.-;130.T.G 0.334 0.136 3581291 3148 2.-.A;72.-.C 0.331 0.300 1251375 3149 -15.T.G80.A.- 0.331 0.238 8128320 3150 75.-.C;127.T.C 0.329 0.315 8356949 3151 87.-.G;118.T.G 0.329 0.277 8552259 3152 75.C.-;86.C.- 0.329 0.275 830221 3153 -21.C.A;74.-.T 0.378 0.279 2820364 3154 0.T.-;2.A.C;18.C.T 0.328 0.303 15456319 3155 -30.C.G;76.-.T 0.328 0.240 8470089 3156 78.-.C;126.C.A 0.328 0.285 8161135 3157 79.G.-;133.A.C 0.327 0.249 8481813 3158 78.A.-;119.C.T 0.327 0.263 2684845 3159 0.T.-;2.A.C;126.C.T 0.326 0.269 8128793 3160 75.-.C;126.C.T 0.326 0.245 15405296 3161 -30.C.G;0.T.- 0.325 0.303 8595845 3162 70.-.T;129.C.A 0.374 0.292 8105737 3163 76.GG.-A;131.A.C;133.A.C 0.323 0.215 8470189 3164 78.-.C;129.C.A 0.323 0.298 14245594 3165 -24.G.T;80.A.- 0.323 0.259 1251224 3166 -15.T.G;81.GA.-T 0.323 0.237 7939926 3167 65.G.-;76.G.- 0.322 0.229 8648998 3168 65.G.T;76.G.- 0.322 0.165 14098317 3169 -20.A.C;2.A.- 0.321 0.261 8032447 3170 72.-.C;131.A.C 0.320 0.251 8061102 3171 74.T.-;76.G.C 0.370 0.180 8481588 3172 78.A.-;120.C.T 0.320 0.267 8565286 3173 75.CG.-T;130.T.C 0.320 0.300 14245896 3114 -24.G.T;76.-.G 0.319 0.198 8066445 3175 74.T.-;127.T.C 0.319 0.230 8150200 3176 77.-.A;129.C.A 0.318 0.223 8479230 3177 78.A.-;118.T.G 0.316 0.213 8482576 3178 78.A.-;113.A.C 0.314 0.236 2271423 3179 0.T.-;123.A.C 0.313 0.263 13907909 3180 -14.A.G;0.T.-;2.A.C 0.313 0.242 8066743 3181 74.T.-;131.A.C;133.A.C 0.312 0.214 8352697 3182 86.C.-128.T.G 0.311 0.186 301021 3183 -28.G.C;0.T.-;2.A.C 0.308 0.178 8480313 3184 78.A.-;125.T.G 0.307 0.265 8136771 3185 76.G.-;87.C.A 0.306 0.204 8019966 3186 72.-.A;82.A.- 0.305 0.276 8632613 3187 66.CT.-G;121.C.A 0.305 0.181 8583599 3188 73.AT.-G;88.G.- 0.305 0.282 8475891 3189 78.A.-;88.G.- 0.304 0.243 8567785 3190 75.C.T;77.-.A 0.304 0.161 8448066 3191 80.A.-;129.C.A 0.303 0.215 8136691 3192 76.G.-;86.C.A 0.302 0.196 15059855 3193 -29.A.G;0.T.-;2.A.C;66.CT.-G 0.301 0.258 13171297 3194 -1.G.T;76.-.G 0.300 0.250 8470230 3195 78.-.C;130.T.G 0.300 0.279 8142877 3196 76.G.-;134.G.C 0.299 0.198 555214 3197 -26.T.C;76.G.- 0.298 0.182 446048 3198 -27.C.A;80.A.- 0.298 0.210 8436528 3199 81.GA.-T;121.C.A 0.297 0.283 8353141 3200 86.C.-;122.A.C 0.296 0.246 8565426 3201 75.CG-T;131.A.G 0.296 0.236 8132576 3202 75.-.C;89.-.C 0.296 0.216 8092121 3203 75.-.A;116.T.G 0.295 0.277 8633166 3204 66.CT.G;132.G.C 0.295 0.138 8142165 3205 76.G.-;124.T.C 0.295 0.253 2686290 3206 0.T.-;2.A.C;114.G.T 0.295 0.236 8161038 3207 79.G.-;129.C.A 0.293 0.266 13853578 3208 -14.A.C;76.-.T 0.293 0.239 807836 3209 -21.C.A;1.TA.-- 0.292 0.265 8469754 3210 78.-.C;119.C.A 0.291 0.158 8137474 3211 76.G.-;101.C.A 0.291 0.226 8160587 3212 79.G.-;120.C.A 0.290 0.161 8142955 3213 76.G.-;131.AGA.CCC 0.29C 0.156 8762708 3214 56.G.T;75.-.G 0.289 0.245 14635887 3215 0.T.-;-29.A.C;75.-.G 0.288 0.221 15455571 3216 -30.C.G;78.-.C 0.287 0.151 8066265 3217 74.T.-;124.T.G 0.285 0.185 8436842 3218 81.GA.-T;130.T.G 0.283 0.228 13846354 3219 -14.A.C;79.G.- 0.282 0.195 8490993 3220 76.-.G;121.C.T 0.281 0.238 14646258 3221 -29.A.C;0.T.-;2.A.C.87.-.T 0.281 0.281 8431378 3222 82.AA.-T;120.C.A 0.279 0.217 8431703 3223 82.AA.-T;126.C.A 0.279 0.249 447910 3224 -27.C.A;73.AT.-G 0.279 0.215 8066683 3225 74.T.-;130.--T.TAG;133.A.G 0.779 0.236 2760011 3226 0.T.-;2.A.C;58.G.T 0.278 0.250 3012063 3227 1.TA.--;123.A.C 0.278 0.271 13855018 3228 -14.A.C;73.A.- 0.277 0.240 8447252 3229 80.A.-;119.C.A 0.277 0.261 8489127 3230 76.-.G;118.T.G 0.776 0.269 8526408 3231 76.-.T;126.C.A 0.775 0.187 8446211 3232 80.A.-;115.T.G 0.273 0.177 8431937 3233 82.AA.-T;133.A.C 0.272 0.216 6558231 3234 18.C.A;73.A.- 0.271 0.209 8159873 3235 79.G.-;115.T.G 0.271 0.220 8602463 3236 73.A.-;119.C.A 0.268 0.230 2684642 3237 0.T.-;2.A.C;131.AGA.CCC 0.268 0.194 8143095 3238 76.G.-.126.C.G 0.266 0.206 1042210 3239 -17.C.A;79.G.- 0.264 0.153 15452123 3240 -30.C.G;88.G.- 0.263 0.246 13852053 3241 -14.A.C;80.A.- 0.262 0.238 8435985 3242 81.GA.-T;115.T.G 0.262 0.210 223220 3243 -30.C.A;76.G.- 0.261 0.213 12148242 3244 2.A.-;124.T.C. 0.260 0.232 8602984 3245 73.A.-;127.T.G 0.259 0.174 318643 3246 -28.G.C;75.-.C 0.259 0.254 15451555 3247 -30.C.G;79.G.- 0.259 0.228 8436802 3248 81.GA.-T;129.C.A. 0.258 0.221 8512529 3249 76.G.-;78.A.T;131.A.C 0.257 0.192 8519060 3250 76.GG.-T;124.T.G 0.255 0.178 1045581 3251 -17.C.A;78.-.C 0.254 0.161 13844608 3252 -14.A.C;74.T.- 0.252 0.231 13171509 3253 -1.G.T;76.GG.-T 0.251 0.179 8336250 3254 89.-.C;121.C.A 0.248 0.177 15455277 3255 -30.C.G;80.A.- 0.246 0.216 8353027 3256 86.C.-;123.A.C 0.246 0.146 8161013 3257 79.G.-;128.T.G 0.245 0.184 8105760 3258 76.GG.-A;129.C.G 0.244 0.201 8558713 3259 74.-.T;123.A.C 0.243 0.218 2681904 3260 0.T.-;2.A.C;116.T.C 0.243 0.228 8558310 3261 74.-.T;127.T.C 0.239 0.165 2684449 3262 0.T.-;2.A.C;130.T.C;132.G.C 0.235 0.191 15052207 3263 -29.A.G;0.T.-;75.-.G 0.233 0.279 8524468 3264 76.G.T;78.A.- 0.232 0.184 7490514 3265 36.C.A;76.GG.-A 0.231 0.201 8633217 3266 66.CT.-G132.G.T 0.275 0.188 8069615 3267 74.T.-;89.-.C 0.224 0.182 15451403 3268 -30.C.G;77.-.A 0.224 0.142 8520167 3269 76.GG.-T;119.C.T 0.222 0.182 10994911 3270 8.G.T;76.G.- 0.222 0.186 2272784 3271 0.T.-;113.A.G 0.218 0.188 8100983 3272 75.C.A;87.-.G 0.209 0.207 13851721 3273 -14.A.C;82.AA.-T 0.209 0.191 8084086 3274 74.-.G;130.T.C 0.207 0.200 8564034 3275 75.CG.-T;116.T.G 0.206 0.195 1117838 3276 -16.C.A;75.CG.-T 0.205 0.200 14023671 3277 -19.G.T;76.GG.-T 0.205 0.189 8519544 3278 76.GG.-T;131.A.C;133.A.C 0.201 0.159 8633185 3279 66.CT.-G 0.200 0.137 14817545 3280 -29.A.C;66.CT.-G 0.199 0.147 1482006 3281 -9.T.C;76.G.- 0.199 0.183 14524849 3282 -28.G.T;75.-.C 0.198 0.181 8470132 3283 78-.C;127.T.G 0.197 0.192 7738954 3284 51.C.A;76.G.- 0.189 0.175 1247296 3285 -15.T.G;79.G.- 0.189 0.163 8519864 3286 76.GG.-T;122.A.G 0.188 0.125 1117512 3287 -16.C.A;76.GG.-T 0.185 0.166 15171788 3288 -29.A.G;66.CT.-G 0.184 0.119 8601732 3289 73.A.-115.T.G 0.183 0.174 6556220 3290 18.C.A;86.C.- 0.182 0.124 8633071 3291 66.CT.-G;129.C.A 0.175 0.164 8499488 3292 78.A.-;80.A.G 0.171 0.166 8519321 3293 76.GG.-T;128.T.C 0.169 0.133 14348190 3294 -25A.C.;86.C.- 0.165 0.107 321013 3295 -28.G.C;74.-.T 0.164 0.163

Approximately 140 modified gRNAs were generated, some by DME and some by targeted engineering, and assayed for their ability to disrupt expression of a target GFP reporter construct by creation of indels. Sequences for these gRNA variants are shown in Table 2. These modified gRNAs exclude modifications to the spacer region, and instead comprise different modified scaffolds (the portion of the sgRNA that interacts with the CRISPR protein). gRNA scaffolds generated by DME include one or more deletions, substitutions, and insertions, which can consist of a single or several base pairs. The remaining gRNA variants were rationally engineered based on knowledge of thermostable RNA structures, and are either terminal fusions of ribozymes or insertions of highly stable stem loop sequences. Additional gRNAs were generated by combining gRNA variants. The results for select gRNA variants are shown in

TABLE 5 Ability of select gRNA variants to disrupt GFP expression Normalized Editing SEQ ID Activity (ave, 2) NO: NAME (Description) spacers n = 6) Std. dev.   5 X2 reference — — 2101 phage replication stable 1.42 0.22 2102 Kissing loop_b1 1.17 0.11 2103 Kissing loop_a 1.18 0.03 2104 32, uvsX hairpin 1.89 0.11 2105 PP7 1.08 0.04 2106 64, trip mut, extended stem truncation 1.69 0.18 2107 hyperstable tetraloop 1.36 0.11 2108 C18G 1.22 0.42 2109 T17G 1.27 0.04 2110 CUUCGG loop 1.24 0.22 2111 MS2 1.12 0.25 2112 −1, A2G, −78, G77T 1.00 0.18 2113 QB 1.44 0.25 2114 45,44 hairpin 0.24 0.41 2115 U1A 1.02 0.05 2116 A14C, T17G 0.86 0.01 2117 CUUCGG loop modified 0.75 0.04 2118 Kissing loop_b2 0.99 0.06 2119 −76:78, −83:87 0.97 0.01 7120 −4 0.93 0.03 2121 extended stem truncation 0.73 0.02 2124 −98:100 0.66 0.05 2125 −1:5 0.45 0.05 2176 −2163 0.57 0.07 2127 =+628, A82T, −84, 0.56 0.04 2128 =+51T 0.52 0.03 2129 −1:4, +G5A, +G86, 0.09 0.21 2130 2174 0.34 0.09 2131 +g72 0.34 0.24 2132 shorten front, CUUCGG loop modified. extend 0.65 0.02 extended 2133 A14C 0.37 0.03 2134 −1:3, +G3 0.45 0.16 2135 =+C45, +T46 0.42 0.04 2136 CUUCGG loop modified, fun start 0.38 0.03 2137 −74:75 0.18 0.04 2138 {circumflex over ( )}T45 0.21 0.05 2139 −69, −94 0.24 0.09 2140 −94 0.01 0.01 2141 modified CUUCGG, minus T in 1st triplex 0.04 0.03 2142 −1:4, +C4, A14C, T17G, +G72, −76:78, −83:87 0.16 0.03 2143 TIC, −73 0.06 0.06 2144 Scaffold uuCG, stem uuCG, Stem swap, t shorten 0.01 0.09 2145 Scaffold uuCG, stem uuCG, Stem swap 0.04 0.03 2146 0.0090408 0.06 0.04 2147 no stem Scaffold uuCG −0.11 0.02 2148 no stem Scaffold uuCG, fun start −0.06 0.02 2149 Scaffold uuCG, stem uuCG, fun start −0.02 0.02 2150 Pseudoknots −0.01 0.01 2151 Scaffold uuCG, stem uuCG −0.05 0.01 2152 Scaffold uuCG, stem uuCG, no start −0.04 0.07 2153 Scaffold uuCG −0.12 0.07 2154 +GCTC36 −0.20 0.05 2155 G quadriplex telomere basket + ends −0.21 0.02 2156 G quadriplex M3q −0.25 0.04 2157 G quadriplex telomere basket no ends −0.17 0.04 2159 Sarcin-ricin loop 0.40 0.03 2160 uvsX, C18G 1.94 0.06 2161 truncated stem loop, C18G, trip mut (T10C) 1.97 0.16 2162 short phage rep, C18G 1.91 0.17 2163 phage rep loop, C18G 1.72 0.13 2164 +G18, stacked onto 64 1.44 0.08 2165 truncated stem loop, C18G, −1 A2G 1.63 0.40 2166 phage rep loop, C18G, trip mut (T10C) 1.76 0.12 2167 short phage rep, Cl8G, trip mut (TUC) 1.20 0.09 2168 uvsX, trip mut (T10C) 1.54 0.12 2169 truncated stem loop 1.50 0.10 2170 +A17, stacked onto 64 1.54 0.13 2171 3′ HDV gnomic ribozyme 1.13 0.13 2172 phage rep loop, trip mut (T10C) 1.39 0.10 2173 −79:80 1.33 0.05 2174 short phage rep, trip mut (T10C) 1.19 0.10 2175 extra truncated stem loop 1.08 0.05 2176 T17G, C18G 0.94 0.09 2177 short phage rep 1.11 0.05 2178 uvsX, C18G, −1b A2G 0.62 0.08 2179 uvsX, C18G, trip mut (T10C), −1 A2G, HDV −99 1.06 0.08 G65T 2180 3′ HDV antigenomic ribozyme 1.20 0.07 2181 uvsX, C18G, trip mut (T10C), −1 A2G, HDV 0.95 0.03 AA(98:99)C 2182 3′ HDV ribozyme (Lior Nissim, Timothy Lu 1.08 0.01 2183 TAC(1:3)GA, stacked onto 64 0.92 0.04 2184 uvsX, −1 A2G 1.46 0.13 2185 truncated stem loop, C18G, trip mut (T10C), −1 0.80 0.02 A2G, HDV −99 G65T 2186 short phage rep, C18G, trip mut (T10C), −1 A2G, 0.80 0.05 HDV −99 G651 2187 3′ sTRSV WT viral Hammerhead ribozyme 0.98 0.03 2188 short phage rep, C18G, −1 A2G 1.78 0.18 2189 short phage rep, C18G, trip mut (T10C), −1 A2G, 0.81 0.08 3′ genomic HDV 2190 phage rep loop, C18G, trip mut (T10C), −1 A2G, 0.86 0.07 HDV −99 G65T 2191 3′ HDV ribozyme (Owen Ryan, Jamie Cate) 0.78 0.04 2192 phage rep loop, C18G, −1 A2G 0.70 0.08 2193 {circumflex over ( )}C55 0.78 0.03 2194 −78, G77T 0.73 0.07 2195 {circumflex over ( )}G1 0.73 0.10 2196 short phage rep, −1 A2G 0.66 0.11 2197 truncated stem loop, C18G, trip mut (T10C), −1 0.68 0.09 A2G 2198 −1, A2G 0.54 0.07 2199 truncated stem loop, trip mut (T10C), −1 A2G 0.40 0.03 2200 uvsX, C18G, trip mut (T10C), −1 A2G 0.35 0.11 2201 phage rep loop, −1 A2G 0.96 0.05 2202 phage rep loop, trip mut (T10C), −1 A2G 0.49 0.06 2203 phase rep loop, C18G, trip mut (T10C), −1 A2G 0.73 0.13 2204 truncated stem loop, C18G 0.59 0.02 2205 uvsX, trip mut (T10C), −1 A2G 0.56 0.08 2206 truncated stem loop, −1 A2G 0.89 0.07 2207 short phage rep, trip mut (T10C), −1 A2G 0.37 0.12 2208 5′HDV ribozme (Owen Ryan, Jamie Cate) 0.39 0.03 2209 5′HDV genomic ribozyme 0.35 0.06 2210 truncated stem loop, C18G, trip mut (T10C), −1 0.24 0.04 A2G, HDV AA(98:99)C 2211 5′env25 pistol ribozyme(with an added 0.33 0.07 CUUCGG loop) 2212 5′HDV antigenomic ribozyme 0.17 0.01 2213 3′ Hammerhead ribozyme (Lior Nissim, Timothy 0.09 0.02 Lu) guide scaffold scar 2214 +A27, stacked onto 64 0.03 0.03 2215 5′Hammerhead ribozyme (Lior Nissim, Timothy 0.18 0.03 Lu) smaller scar 2216 phage rep loop, C18G, trip mut (T10C), −1 A2G, 0.13 0.04 HDV AA(98:99)C 2217 −27, stacked onto 64 0.00 0.03 2218 3′ Hatchet 0.09 0.01 2219 3′ Hammerhead ribozyme (Lior Nissim, Timothy 0.05 0.03 Lu) 7220 5′Hatchet 0.04 0.03 2221 5′HDV ribozyme (Lior Nissitn, Timothy Lu) 0.08 0.01 7222 5′Hammerhead ribozyme (Lior Nissim, Timothy 0.22 0.01 Lu) 2223 3′ HH15 Minimal Hammerhead ribozyme 0.01 0.01 2224 5′ RBMX recruiting motif −0.08 0.03 7225 3′ Hammerhead ribozyme (Lior Nissim, Timothy −0.04 0.02 Lu) smaller scar 2226 3′ env25 pistol ribozyme(with an added −0.01 0.01 CUUCGG loop) 2227 3′ Env-9 Twister −0.17 0.02 2228 +ATTATCTCATTACT25 −0.18 0.27 7229 5′Env-9 Twister −0.02 0.01 2230 3′ Twisted Sister 1 −0.27 0.02 7231 no stem −0.15 0.03 2232 5′HH15 Minimal Hammerhead ribozyme −0.18 0.04 2233 5′Hammerhead ribozyme (Lior Nissim, Timothy −0.14 0.01 Lu) guide scaffold scar 2234 5′Twisted Sister 1 −0.14 0.04 7235 5′sTRSV WT viral Hammerhead ribozyme −0.15 0.02 2236 148, =+G55, stacked onto 64 3.40 0.18 2239 175, trip mut, extended stem truncation, with [T] 1.18 0.09 deletion at 5′ end

Although guide stability can be measured thermodynamically (for example, by analyzing melting temperatures) or kinetically (for example, using optical tweezers to measure folding strength), without wishing to be bound by any theory it is believed that a more stable sgRNA bolsters CRISPR editing efficiency. Thus, editing efficiency was used as the primary assay for improved guide function.

The activity of the gRNA scaffold variants was assayed using E6 and E7 spacers as described above, targeting GFP. The starting sgRNA scaffold in this case was a reference Planctomyces CasX tracr RNA fused to a Planctomyces crispr RNA (crRNA) using a “GAAA” stem loop (SEQ ID NO: 5). This sgRNA scaffold was used a base for DME and rationally engineered mutations. The activity of variant gRNAs shown in Table 6 was normalized to the activity of this starting, or base, sgRNA scaffold.

The sgRNA scaffold was cloned into a small (less than 3 kilobase pair) plasmid with a 3′ type II restriction enzyme site for dropping in different spacers. The spacer region of the sgRNA is the part of the sgRNA interacts with the target DNA, and does not interact directly with the CasX protein. Thus, scaffold engineering should be spacer independent. One way to achieve this is by executing sgRNA DME and testing engineered sgRNA variants using several distinct spacers, such as the E6 and E7 spacers targeting GFP. This reduces the possibility of creating an sgRNA scaffold variant that works well with one spacer sequence targeting one genetic target, but not other spacer sequences directed to other targets. For the data shown in Table, 6, the E6 and E7 spacer sequences targeting GFP were used. Repression of GFP expression by sgRNA variants was normalized to GFP repression by the sgRNA starting scaffold of SEQ ID NO: 5 assayed with the same spacer sequence(s).

Activity of select sgRNA variants generated by DME and rational engineering is shown in FIGS. 5A-5E, mean change in activity is shown in Table 6, and sgRNA variant sequences are provided in Table 2. sgRNA variants with increased activity were tested in HEK293 cells as described in Example 1. FIG. 5C shows that select sgRNA variant have improved GFP editing when assayed in HEK293 cells. FIG. 5D shows that in some cases, activity can be improved by appending ribozyme sequences. FIG. 5E shows that sgRNA variants comprising combinations of changes, for example those generated by DME or replacing stem loop sequences, can further improve editing activity.

Example 4: Mutagenesis of CasX Protein Produces Improved Variants

A selectable, mammalian-expression plasmid was constructed that included a reference, also referred to herein as starting or base, CasX protein sequence, an sgRNA scaffold, and a destination sequence that can be replaced by spacer sequences. In this case, the starting CasX protein was Stx2 (SEQ ID NO: 2), the wild type Planctomycetes CasX sequence and the scaffold was the wild type sgRNA scaffold of SEQ ID NO: 5. This destination plasmid was digested using the appropriate restriction enzyme following manufacturer's protocol. Following digestion, the digested DNA was purified using column purification according to manufacturer's protocol. The E6 and E7 spacer oligos targeting GFP were annealed in 10 uL of annealing buffer. The annealed oligos were ligated to the purified digested backbone using a Golden Gate ligation reaction. The Golden Gate ligation product was transformed into chemically competent E. coli bacterial cells and plated onto LB agar plates with the appropriate antibiotic. Individual colonies were picked, and the GFP spacer insertion was verified via Sanger sequencing.

The following methods were used to construct a DME library of CasX protein variants. The functional Plm CasX protein, which is a 978 residue multi-domain protein (SEQ ID NO: 2) can function in a complex with a 108 bp sgRNA scaffold (SEQ ID NO: 5), with an additional 3′ 20 bp variable spacer sequence, which confers DNA binding specificity. Construction of the comprehensive mutation library thus required two methods: one for the protein, and one for the sgRNA. Plasmid recombineering was used to construct a DME protein library of CasX protein variants. PCR-based mutagenesis was used to construct an RNA library of the sgRNA. Importantly, the DME approach can make use of a variety of molecular biology techniques. The techniques used for genetic library construction can be variable, while the design and scope of mutations encompasses the DME method.

In designing DME mutations for the reference CasX protein, synthetic oligonucleotides were constructed as follows: for each codon, three types of oligonucleotides were synthesized. First, the substitution oligonucleotide replaced the three nucleotides of the codon with one of 19 possible alternative codons which code for the 19 possible amino acid mutations. 30 base pair flanking regions of perfect homology to the target gene allow programmable targeting of these mutations. Second, a similar set of 20 synthetic oligonucleotides encoded the insertion of single amino acids. Here, rather than replace the codon, a new region consisting of three base pairs was inserted between the codon and the flanking homology region. Twenty different sets of three nucleotides were inserted, corresponding to new codons for each of the twenty amino acids. Larger insertions can be built identically but will contain an additional three, six, or nine base pairs, encoding all possible combinations of two, three, or four amino acids. Third, an oligonucleotide was designed to remove the three base pairs comprising the codon, thus deleting the amino acid. As above, oligonucleotides can be designed to delete one, two, three, or four amino acids. Plasmid recombineering was then used to recombine these synthetic mutations into a target gene of interest, however other molecular biology methods can be used in its place to accomplish the same goal.

Table 6 shows the fold enrichment of CasX protein variant DME libraries created from the reference protein of SEQ ID NO: 2, which were then subjected to DME selection/screening processes.

In Table 6 below, the read counts associated with each of the listed variants was determined. Each variant was defined by its position (0-indexed), reference base, and alternate base. Only sequences with at least 10 reads (summed) across samples were analyzed, to filter from 457K variants to 60K variants. An insertion at position i indicates an inserted base between position i−1 and i (i.e. before the indicated position). ‘counts’ indicates the sequencing-depth normalized read count per sequence per sample. Technical replicates were combined by taking the geometric mean. ‘log 2enrichment’ gives the median enrichment (using a pseudocount of 10) across each context, or across all samples, after merging for technical replicates. Each context was normalized by its own naive sample. Finally, the ‘log 2enrichment_err’ gives the ‘confidence interval’ on the mean log 2 enrichment. It is the std. deviation of the enrichment across samples*2/sqrt of the number of samples. Below, only the sequences with median log 2enrichment −log 2enrichment_err>0 are shown (60274 sequences examined).

The computational protocol used to generate Table 6 was as follows: each sample library was sequenced on an Illumina HiSeq for 150 cycles paired end (300 cycles total). Reads were trimmed to remove adapter sequences, and aligned to a reference sequence. Reads were filtered if they did not align to the reference, or if the expected number of errors per read was high, given the phred base quality scores. Reads that aligned to the reference sequence, but did not match exactly, were assessed for the protein mutation that gave rise to the mismatch, by aligning the encoded protein sequence of the read to the protein sequence of the reference at the aligned location. Any consecutive variants were grouped into one variant that extended multiple residues. The number of reads that support any given variant was determined for each sample. This raw variant read count per sample was normalized by the total number of reads per sample (after filtering for low expected number of errors per read, given the phred quality scores) to account for different sequencing depths. Technical replicates were combined by finding the geometric mean of variant normalized read count (shown below, ‘counts’). Enrichment was calculated for each sample by diving by the naive read count (with the same context—i.e. D2, D3, DDD). To downweight the enrichment associated with low read count, a pseudocount of 10 was added to the numerator and denominator during the enrichment calculation. The enrichment for each context is the median across the individual gates, and the enrichment overall is the median enrichment across the gates and contexts. Enrichment error is the standard deviation of the log 2 enrichment values, divided by the sqrt of the number of values per variant, multiplied by 2 to make a 95% confidence interval on the mean.

Heat maps of DME variant enrichment for each position of the reference CasX protein are shown in FIGS. 7A-7I and FIGS. 8A-8C. Fold enrichment of DME variants with single substitutions, insertions and deletions of each amino acid of the reference CasX protein of SEQ ID NO: 2 are shown. FIGS. 7A-7I and Table 6 summarize the results when the DME experiment was run at 37° C. FIGS. 8A-8C summarize the results when the same experiment was run at 45° C. A comparison of the data in FIGS. 7A-7I and FIGS. 8A-8C shows that running the same assay at two temperatures enriches for different variants. A comparison of the two temperatures thus indicates which amino acid residues and changes are important for thermostability and folding, and these amino acids can then be targeted to produce CasX protein variants with improved thermostability and folding.

TABLE 6 Fold enrichment of CasX DME Variants Pos. Ref. Alt. Med. Enrich. 95% CI 11 R N 3.123689614 1.666090155 13 — AS 2.772897791 0.812692873 13 — AG 2.740825108 1.138556052 12 — V 2.739405927 1.743064315 13 — TS 2.69239793 1.005397595 12 — Y 2.676525308 1.621386271 754 FE LA 2.638126094 0.709679147 13 — L 2.63160466 1.131924801 14 V S 2.616515776 1.515637887 877 V G 2.558943878 1.132565008 21 — D 2.295527175 0.893253582 12 — PG 2.222956581 1.243693989 824 V M 2.181465681 1.137291381 12 — Q 2.102167857 1.396704669 13 L E 2.049540302 0.886997965 12 R A 2.046419725 1.229773759 889 S K 2.030682939 0.721857305 791 — Q 1.996189679 0.799796529 21 — S 1.907167641 0.736834562 14 — A 1.89090961 1.25865759 11 R M 1.88125645 0.779897343 856 Y R 1.83253552 0.74976479 707 A Q 1.830052571 0.555234229 16 — D 1.826796594 1.168291076 17 S G 1.799890039 0.536675637 931 S M 1.798321904 1.171026479 13 L V 1.782912682 0.513630591 11 — AS 1.782444935 0.75642805 856 Y K 1.748619552 0.651026121 796 — AS 1.742437726 0.859039085 877 V D 1.738762289 0.688664606 459 K W 1.696823829 0.67904004 891 E K 1.6928634 0.819015932 9 — T 1.667698181 0.626564384 19 — R 1.664532235 0.885325268 11 R P 1.655382042 1.234907956 793 — L 1.585086754 0.91714318 931 S L 1.583295371 0.643295534 12 — AG 1.580094246 1.037517499 770 M P 1.577648056 1.061356917 791 L E 1.551380949 0.823309399 21 — A 1.5.42633652 0.760237264 814 F H 1.510927821 0.672796928 12 — C 1.506305374 0.730799624 791 L S 1.505731571 0.598349327 792 — AS 1.474378912 0.833339427 12 — L 1.46896091 0.783746198 795 T — 1.465811841 0.744738295 792 — Q 1.462809015 0.586506727 11 R S 1.459875087 0.740946571 11 R T 1.450818176 0.908088492 738 A V 1.397545277 0.638310372 791 — Y 1.382702158 0.877495368 384 E P 1.36783963 0.775382596 793 — ST 1.351743597 0.608183464 738 A T 1.349932545 0.581386051 781 W Q 1.342276465 0.719454459 17 — G 1.340746587 0.878053267 12 — AS 1.333635165 1.19716917 771 A V 1.292995852 0.871463205 792 — E 1.290525566 1.195462062 921 A M 1.28763891 0.560591034 979 LE[stop]GS- VSSKDL (SEQ ID 1.282505495 0.371661154 NO: 3664) 770 M Q 1.279910431 1.186538897 16 — AG 1.271874994 0.55951096 384 E N 1.247124467 0.607911368 979 L- VS 1.239823793 0.315337927 979 LE[stop] VSS 1.233215135 0.36262523 658 -D APG 1.220851584 0.979760686 979 L-E VSS 1.21568584 0.37106558 385 E S 1.210243487 0.826999735 979 LE[stop]GS-PGIK VSSKDLQASNK 1.208612972 0.286427519 (SEQ ID NO: (SEQ ID NO: 3666) 3665)[stop] 793 — SA 1.192367811 0.72089465 739 R A 1.188987234 0.611670208 795 — AS 1.183930928 0.90542554 979 LE[stop]GS-P VSSKDLQ (SEQ ID 1.180100725 0.35995062 NO: 3667) 977 V K 1.17977084 0.720108501 658 -D AAS 1.173300666 0.50353561 14 — TS 1.173232132 0.700156049 10 — V 1.164019233 1.085055677 375 P K 1.163918709 0.891802018 795 — AG 1.14629929 0.481029275 979 LE[stop]GSPG VSSKDLQ (SEQ ID 1.143633475 0.340695621 (SEQ ID NO: NO: 3667) 3668) 979 LE VS 1.142516835 0.386398408 877 V Q 1.141917178 0.655790093 979 L-E[stop] VSSK (SEQ ID NO: 1.125229136 0.372301096 3669) 936 R Q 1.117866436 0.745233062 979 LE[stop]GS-PGIK VSSKDLOASN (SEQ 1.111969193 0.311410682 (SEQ ID NO: ID NO: 3670) 3665) 396 V Q 1.105278825 0.646150998 979 LE[stop]GSP VSSKDL (SEQ ID 1.104849849 0.260693612 NO: 3664) 353 L F 1.103922948 0.510520582 979 LE[stop]GS-PG VSSKDLQA (SEQ ID 1.100880851 0.345695892 (SEQ ID NO: NO: 3671) 3668) 697 Y H 1.097977697 0.419010874 796 — PG 1.095168865 0.816765224 4 — TS 1.088089915 0.693109756 10 R K 1.085472062 0.382234839 790 G M 1.066566819 0.686227232 921 A K 1.056315246 0.70226115 696 — R 1.049001055 0.880941583 9 L 1.039309233 0.528320595 979 LE[stop]GSPGIK VSSKDLQASNK 1.037884742 0.299531766 (SEQ ID NO: (SEQ ID NO: 3666) 3672)[stop]N 13 — S 1.031062599 0.727357338 384 E R 1.028117481 0.683537724 21 K D 1.019445543 0.748518701 978 [stop] G 1.016498062 0.514955543 979 L-E[stop]G VSSKD (SEQ ID NO: 1.016126075 0.353515679 3673) 10 R N 1.010184099 0.846798556 794 — PG 1.00924007 0.987312969 791 L Q 1.004388299 0.361910793 792 P G 1.002325281 0.805296973 877 V C 0.995089773 0.566724231 476 C Y 0.984546648 0.686487573 19 — PG 0.984071689 0.738694244 979 LE[stop]GSPGI VSSKDLQA (SEQ ID 0.972011014 0.292930615 (SEQ ID NO: NO: 3671) 3674) 752 L P 0.971338521 0.459371253 12 R C 0.969988229 0.745286116 12 R Y 0.962112567 0.714384629 979 LE[stop]GSPGIK VSSKDLQAS (SEQ 0.960035296 0.298173201 (SEQ ID NO: ID NO: 3675) 3672) 18 — PG 0.952532997 0.782330584 778 M I 0.945963409 0.345538178 798 S P 0.942103893 0.470224487 16 D G 0.941159649 0.341870864 22 A Q 0.937573643 0.676316271 754 FE IA 0.935796963 0.660936674 1 Q K 0.935474248 0.373656765 14 V F 0.932689058 0.742246472 8 K I 0.928472117 0.521050669 384 E G 0.920571639 0.452302777 732 D T 0.912254061 0.759438627 658 D Y 0.894131769 0.312165116 211 L P 0.887315174 0.318877781 14 V A 0.885138345 0.699864156 979 LE[stop]G V-S 0.88.4897395 0.252782429 13 — 0.883212774 0.713984249 979 LE[stop]G VSSK (SEQ ID NO: 0.881127427 0.417135617 3669) 386 D K 0.879045429 0.728272074 5 R I 0.871114116 0.317513506 660 — AS 0.862493953 0.798632847 877 V M 0.855677916 0.267740831 741 L W 0.851844349 0.594072278 24 — W 0.835220929 0.745009807 755 E [stop] 0.833955657 0.31600491 928 I T 0.832425124 0.307759846 979 LE[stop]GS-PGI VSSKDLQAS (SEQ 0.822335062 0.317179456 (SEQ ID NO: ID NO: 3675) 3674) 781 W K 0.810589018 0.686153856 791 L R 0.806201856 0.611654466 979 LE[stop]GSPGIK VSSKDLQASN (SEQ 0.80600706 0.220866187 (SEQ ID NO: ID NO: 3670) 3672)[stop] 711 E Q 0.793874739 0.38732268 703 T N 0.791134752 0.735228799 793 S — 0.7821232 0.523699668 385 E K 0.781091846 0.579724424 955 R M 0.780963169 0.340474646 469 — N 0.775656135 0.541879732 788 V T 0.770125047 0.581859138 705 Q R 0.76633283 0.261069709 9 — TS 0.763723778 0.674640849 979 LE[stop]GS VSSKD (SEQ ID NO: 0.761764547 0.205465156 3673) 715 A K 0.761122086 0.540516283 384 E K 0.760859162 0.22641046 591 QG R- 0.757963418 0.374903235 316 R M 0.757086682 0.310302995 770 M T 0.753193128 0.319236781 384 E Q 0.752976137 0.602376709 17 S E 0.752400908 0.414988963 755 E D 0.74863141 0.212934852 12 R — 0.743504623 0.648509511 938 Q E 0.741570425 0.469451701 657 I V 0.73806027 0.256874713 −1 S T 0.735179004 0.144429929 2 E [stop] 0.734071396 0.323713248 384 E A 0.733775595 0.660142332 891 E Y 0.733458673 0.165192765 643 V F 0.732765961 0.577614171 796 — C 0.732364738 0.485790322 280 L M 0.731787266 0.258239226 695 — K 0.730902961 0.509205112 343 W L 0.725824372 0.292120452 3 — IKRINK (SEQ ID NO: 0.721338414 0.470264314 3676) 732 D N 0.71945188 0.416870981 687 — PTH 0.716433371 0.159856315 176 A D 0.71514177 0.206626688 485 W L 0.713411462 0.238105577 22 A D 0.710738042 0.32510753 193 L P 0.709349304 0.242633498 899 R M 0.707875506 0.298429738 886 KG R- 0.706803824 0.286241441 796 — TS 0.697218521 0.492426198 329 P H 0.696817542 0.314817482 273 L P 0.696199602 0.349703999 31 L M 0.696080627 0.331245769 645 — E 0.692307595 0.590013131 9 I Y 0.689813642 0.667593375 9 I N 0.688953393 0.257809633 919 H R 0.688781806 0.363439859 687 P H 0.684782236 0.310607479 332 P H 0.672484781 0.326219913 796 — N 0.672333697 0.64437503 421 W L 0.667702097 0.291970479 875 E [stop] 0.66617872 0.287006304 378 L K 0.664474618 0.393361359 891 E Q 0.663650921 0.312291932 926 L M 0.661737644 0.525550321 656 G C 0.659813316 0.293973226 4 K N 0.656251908 0.302190904 774 Q E 0.654737733 0.134116674 −1 S C 0.652333059 0.118222939 21 — AS 0.651563705 0.48650799 185 L P 0.649897837 0.225081568 38 P T 0.648698083 0.350485275 936 R H 0.648045448 0.423309347 813 G C 0.644003475 0.310838653 786 L M 0.643153738 0.314936636 942 K N 0.639528926 0.249553292 293 Y H 0.636816244 0.207205991 542 F L 0.635949082 0.181128276 303 W L 0.635588216 0.261903568 979 LE V[stop] 0.635165807 0.329009453 578 P H 0.634392073 0.324298942 687 — PT 0.633217575 0.355316701 886 K N 0.632562679 0.231080349 20 K R 0.632186797 0.237509121 248 L P 0.631068881 0.180279623 18 N S 0.630660766 0.266585824 836 M V 0.630065132 0.266534124 116 K N 0.629540403 0.234219411 847 EG GA 0.628295048 0.299740787 912 L P 0.627137425 0.187179246 92 P H 0.626243107 0.350245614 299 Q K 0.623386276 0.302029469 707 A T 0.622086487 0.275515174 669 L M 0.620453868 0.351072046 789 E D 0.617920878 0.216264385 916 F S 0.617302977 0.309372822 55 P H 0.616365993 0.329695842 936 R G 0.615282844 0.189389227 595 F L 0.615176885 0.154670433 0 M I 0.612039515 0.303853593 381 L R 0.609889042 0.420808291 945 T A 0.609683347 0.258353939 389 K N 0.609647876 0.274048697 755 E G 0.607714844 0.078377344 559 I M 0.606040482 0.27336203 825 L P 0.604240507 0.192490062 733 M T 0.603960776 0.340233556 664 P T 0.60370266 0.234348448 10 R T 0.602483957 0.372156893 964 F L 0.60175279 0.17004436 911 C S 0.601303891 0.279730674 788 Y G 0.600935917 0.580949772 447 Q K 0.600543047 0.297568309 13 L P 0.599989903 0.236688663 193 L M 0.599332216 0.309308194 114 P H 0.599262194 0.344450733 660 G R 0.599221963 0.319640645 894 S T 0.599084973 0.166490359 904 P H 0.59783828 0.349.499416 782 L T 0.595786463 0.513346845 944 Q K 0.595243666 0.351818545 207 P H 0.595218482 0.277632613 151 H N 0.595188624 0.277503327 495 A K 0.594637604 0.315764586 −1 S P 0.594582952 0.377333364 480 L E 0.594055289 0.432259346 469 E A 0.594025118 0.30338267 11 R G 0.59320688 0.163279008 85 W L 0.591691074 0.2708118 15 K E 0.587925122 0.149546484 755 E K 0.586636571 0.217538569 337 Q R 0.585098232 0.172195554 877 V A 0.584567684 0.258968272 793 — TS 0.583269098 0.45091329 670 T R 0.582033902 0.112618756 925 A P 0.581907283 0.186614282 659 R L 0.580864225 0.319384189 306 L P 0.578183307 0.210431982 676 P Q 0.577757554 0.308473522 877 V E 0.57724394 0.294796776 19 T A 0.576889973 0.198407278 14 V D 0.574902804 0.437270334 887 G Q 0.574717855 0.519529758 935 L V 0.573813105 0.185021716 961 W L 0.573698555 0.253700288 23 — GP 0.572198674 0.570313308 541 R L 0.571508027 0.254421711 288 E D 0.571482463 0.24542675 742 L V 0.570384839 0.3027928 931 S T 0.570369019 0.120673525 623 — RRTRQDE (SEQ ID 0.569913903 0.141118873 NO: 3677) 27 P H 0.569605452 0.285015385 28 M T 0.56885021 0.216863369 907 E [stop] 0.567613159 0.345163987 577 D Y 0.567493308 0.253952459 672 P H 0.566921749 0.31335168 669 L P 0.564276636 0.224594167 52 E D 0.564250133 0.246311739 46 N T 0.563094073 0.208662987 5 R G 0.560139309 0.15069426 912 L V 0.559515875 0.111973397 40 L M 0.558605774 0.239058063 923 Q [stop] 0.558515774 0.34688202 979 L-E[stop]G VSSKE (SEQ ID NO: 0.557263947 0.22994802 3678) 41 R T 0.555902565 0.199937528 179 E [stop] 0.555817911 0.245362937 344 W L 0.555474112 0.286390208 63 R M 0.554978749 0.336590825 1 Q R 0.554755158 0.207724233 9 I V 0.554053334 0.219348804 914 C [stop] 0.552658801 0.347714953 836 M I 0.551813626 0.180327214 856 Y H 0.549262192 0.369311354 620 L M 0.548957556 0.322210662 926 L P 0.547714601 0.450095044 377 L P 0.546553821 0.20366425 920 A S 0.545992524 0.484867291 961 W [stop] 0.544371204 0.244581668 746 V G 0.543151726 0.512718498 554 — RFY 0.542549772 0.20487223 664 P H 0.542466431 0.281534858 5 R [stop] 0.541304946 0.166704906 803 Q K 0.540975244 0.291121648 652 M I 0.540953074 0.217563311 326 KG R- 0.540593574 0.402287668 789 E [stop] 0.540122225 0.236046287 889 S L 0.539927241 0.375365013 10 R I 0.539433301 0.326816988 725 K N 0.539088606 0.178127049 603 L P 0.538897648 0.229282796 15 K R 0.538786311 0.154390287 541 R G 0.537572295 0.133876643 632 L M 0.537440995 0.246129141 665 A S 0.536996011 0.286216687 650 K E 0.536939626 0.139863469 932 W L 0.536075206 0.314946873 684 L M 0.535519584 0.338883641 918 I R 0.535067274 0.304580877 10 R G 0.534873359 0.3557865 575 F L 0.534865272 0.139851134 737 I G 0.534759369 0.303617666 907 E G 0.534688762 0.240107856 703 T R 0.53396819 0.160757401 962 Q E 0.533896042 0.302336405 764 Q H 0.53385913 0.24340782 793 S T 0.533306619 0.17379091 6 I M 0.533192185 0.188523563 467 L P 0.533022246 0.179464215 244 Q [stop] 0.532045714 0.262393061 8 K N 0.531704561 0.294399975 508 F V 0.529042378 0.192146822 665 A P 0.529013767 0.174049723 46 NL T[stop] 0.529006897 0.272198259 3 I V 0.528916598 0.14506718 518 W S 0.528332889 0.199792834 792 P A 0.528028079 0.112407207 13 L A 0.526728857 0.318983292 56 Q K 0.526387006 0.188452852 878 N S 0.526073971 0.27887921 213 Q E 0.525578421 0.16885346 748 Q H 0.525406412 0.200108279 15 K N 0.525094369 0.273038164 954 K N 0.524763966 0.208680978 835 W L 0.524725836 0.26540236 847 E D 0.524019387 0.23897504 608 L M 0.523890883 0.248052068 932 W R 0.523129128 0.299781077 21 K N 0.522953217 0.250998038 790 G [stop] 0.5229473 0.262740975 707 A D 0.522560362 0.214610237 954 K V 0.522546614 0.349200627 952 T A 0.521534511 0.149679645 892 A D 0.521298872 0.228218092 847 — EGQITYY (SEQ ID 0.521149636 0.115331328 NO: 3679) 7 N I 0.521103862 0.202836314 702 R M 0.520743818 0.247227864 901 S G 0.520379757 0.143482219 560 N H 0.519240936 0.286066696 350 V M 0.518159753 0.277778553 535 F L 0.518099748 0.153008763 512 Y H 0.517168474 0.223506594 278 I M 0.516794992 0.238648894 746 V A 0.51672383 0.202625874 664 P R 0.516702968 0.252959416 −1 S A 0.516689693 0.142459137 298 A D 0.51645727 0.257163483 361 G C 0.515521808 0.242033529 424 I V 0.515355817 0.185117148 907 E D 0.514835248 0.277377403 923 Q E 0.514826301 0.324456465 413 W L 0.514728329 0.241932097 748 Q R 0.514571576 0.240563892 591 Q H 0.514415886 0.331792035 1 Q E 0.514404075 0.263908964 171 P T 0.513803013 0.237477165 544 K R 0.512919851 0.163480182 677 — LSRFKD (SEQ ID 0.511837147 0.194279796 NO: 3680) 377 L M 0.511718619 0.274965484 1 Q H 0.511496323 0.29357307 202 R M 0.511365875 0.303187834 422 E [stop] 0.511043687 0.224103239 922 E [stop] 0.510570886 0.450135707 407 — KKHGED (SEQ ID 0.510425363 0.211479415 NO: 3681) 8 K A 0.510125467 0.417426274 300 I M 0.510084254 0.178542003 668 A P 0.509985424 0.202934866 917 E K 0.509268127 0.386629094 12 R I 0.509210198 0.267908359 326 K N 0.508325806 0.277854988 802 A W 0.507146644 0.398619961 627 Q H 0.506946344 0.17779761 705 Q K 0.506601342 0.205329495 935 L P 0.505173269 0.279127846 636 L P 0.504912592 0.279575261 378 L V 0.504856105 0.146721248 770 M I 0.502407214 0.148647414 302 I I 0.502263164 0.328365742 584 P H 0.501836401 0.188263444 962 Q H 0.501557133 0.21210836 909 F L 0.501216251 0.397907118 522 G C 0.50035512 0.232143601 233 M I 0.500272986 0.246898577 284 P R 0.499965267 0.18413971 639 E D 0.499845638 0.16815712 351 K E 0.49917291 0.274793088 12 R S 0.498984129 0.193129295 920 A V 0.498509984 0.394258252 709 E [stop] 0.498173203 0.222297538 443 S H 0.498010803 0.445232627 27 P L 0.497724007 0.373177387 849 Q K 0.497661989 0.259123161 793 — Q 0.497102388 0.47673495 750 A G 0.496799617 0.243940432 26 G C 0.496365725 0.228107532 706 A D 0.494947511 0.225683587 431 L P 0.494543065 0.192514906 13 LV AS 0.494489513 0.367074627 0 M V 0.49405414 0.206071479 614 R I 0.494053835 0.209299062 248 L M 0.49299868 0.24880607 81 L M 0.492127571 0.369172142 418 — D 0.49144742 0.21486801 914 C R 0.490784001 0.353820866 3 I S 0.490305334 0.219289736 781 W L 0.490256264 0.225567162 234 G [stop] 0.489800943 0.231905474 369 A V 0.489746571 0.142680124 685 G C 0.48966455 0.174412352 498 A S 0.489397172 0.173872708 746 V D 0.488692506 0.484120982 666 — AG 0.488446913 0.383322789 309 W L 0.487964134 0.209151088 979 — VSSK (SEQ ID NO: 0.486810051 0.287650542 3669) 27 P R 0.486771244 0.185539954 583 L M 0.486474099 0.232216764 760 G R 0.485722591 0.195838563 596 I T 0.485474246 0.130718203 189 G [stop] 0.484957086 0.271997616 884 W L 0.48469466 0.210361106 162 E [stop] 0.484515492 0.270313618 405 L P 0.484058533 0.143471721 815 T A 0.483688268 0.140346764 875 E D 0.483680843 0.230122106 703 T K 0.483561705 0.243688021 35 V A 0.48268809 0.163074127 320 K E 0.482629615 0.202594011 203 E D 0.482289135 0.173584261 202 R S 0.482184999 0.1640178 613 G C 0.482001189 0.220237462 220 A P 0.481251117 0.159715468 920 A G 0.481026982 0.321704418 874 E Q 0.480905869 0.250463545 192 A G 0.480770514 0.112319124 578 P T 0.48002354 0.203348553 515 A P 0.480000762 0.142980394 921 D Y 0.479522102 0.330930172 17 S R 0.479410291 0.242870401 23 G C 0.47738757 0.286426817 892 A G 0.477302415 0.253000116 832 A T 0.47606534 0.23451824 421 W [stop] 0.475666945 0.216973062 316 R S 0.47464939 0.264534919 681 K N 0.474468269 0.192816933 22 A V 0.474221933 0.206217506 691 L M 0.473867575 0.189071763 95 L V 0.473859579 0.188485586 827 K N 0.47365473 0.198868181 858 R M 0.473407136 0.257236194 519 Q P 0.472315609 0.224391717 95 L P 0.471361064 0.162277972 976 A T 0.470889659 0.109031 782 L I 0.470558203 0.125178365 723 A S 0.469929973 0.218713854 24 K R 0.469399175 0.236250784 748 Q E 0.46890075 0.291020418 686 — NPT 0.468711675 0.157459195 1 Q L 0.468380179 0.341181409 466 G V 0.467982153 0.207162352 346 — MVC 0.467747954 0.140593808 746 V L 0.467699466 0.162488099 101 Q K 0.467562845 0.263058522 99 V L 0.467355555 0.098627209 354 I M 0.46704321 0.243813968 826 E [stop] 0.466802563 0.164892155 150 P L 0.466773068 0.200507693 476 C R 0.466682009 0.123054893 38 P H 0.466309116 0.291701454 120 E [stop] 0.465867266 0.21730484 370 D R 0.465477814 0.252126933 7 N K 0.465102103 0.221573061 55 P T 0.465075846 0.236340763 681 K E 0.464515385 0.142005053 781 W C 0.464133122 0.295451154 946 N D 0.463522655 0.373105851 368 L M 0.463023353 0.266615533 0 M T 0.462868938 0.232012879 737 T A 0.462760296 0.301960654 847 — EGQI (SEQ ID NO: 0.462759431 0.219565444 3682) 0 M K 0.462242932 0.245616902 711 E [stop] 0.461879161 0.191719959 357 K N 0.461332764 0.184353442 434 H D 0.461154018 0.191223379 910 V E 0.460870605 0.281013173 922 E D 0.460080408 0.286351122 480 L D 0.459795711 0.404684507 772 E G 0.459510918 0.312503946 369 A P 0.459368992 0.154954523 148 G C 0.459321913 0.21989387 565 E [stop] 0.459284191 0.257970072 472 K N 0.458126194 0.217353923 19 T K 0.458002489 0.250652905 550 F L 0.457885561 0.135416611 612 E D 0.457477443 0.18018994 761 F L 0.457399802 0.126293846 104 P H 0.457206235 0.205670388 588 G C 0.457151433 0.254991865 516 F L 0.456927783 0.127509134 147 K N 0.456444496 0.280029247 651 P H 0.456356549 0.186081926 2 E D 0.456056175 0.35763481 643 V G 0.455368156 0.295796806 524 K N 0.45482233 0.143701874 18 N K 0.454706199 0.199478283 5 R T 0.45449471 0.277079709 920 A P 0.45449471 0.288443793 701 Q H 0.453812486 0.146230302 891 E [stop] 0.453785945 0.233457013 133 C W 0.453639333 0.137105208 370 G V 0.453597184 0.202403506 548 E D 0.453077345 0.109679349 689 H D 0.453055551 0.09160837 931 S R 0.45302365 0.382294772 133 C [stop] 0.452586533 0.10138833 868 E [stop] 0.452282618 0.301898798 33 V L 0.451975838 0.159872004 266 D Y 0.451699485 0.165335876 197 E D 0.451539434 0.154482619 661 E [stop] 0.45138977 0.234896635 897 K N 0.451376493 0.172130787 894 S G 0.451201568 0.216541569 46 N K 0.450854268 0.293319843 42 E [stop] 0.450047213 0.226279727 20 K N 0.449773662 0.196721642 285 H N 0.44861581 0.243329874 47 L V 0.448153393 0.267732388 953 D E 0.448187279 0.183598076 8 K E 0.447865624 0.173510738 255 K N 0.447654062 0.257753112 965 Y [stop] 0.447638184 0.206848878 381 L V 0.447518148 0.24623578 938 Q K 0.44750144 0.297903846 719 S C 0.4472033 0.232249869 89 Q K 0.447094951 0.222907496 735 R L 0.447058488 0.220193339 673 E G 0.446968171 0.213951556 126 G C 0.446802066 0.204738022 919 H D 0.446668628 0.327432207 23 G V 0.446595867 0.2102612 733 M I 0.446594817 0.174646778 310 Q E 0.446297431 0.123671296 729 L V 0.445993097 0.433135394 455 W L 0.445597501 0.281894997 215 G V 0.445352945 0.205217458 135 P T 0.44528202 0.217449002 936 R I 0.445259832 0.32221387 519 Q K 0.444720886 0.28933765 656 G R 0.444552088 0.279063867 613 G R 0.444378039 0.117584873 16 D Y 0.44433236 0.241975919 5 R K 0.443724261 0.262708705 3 I M 0.443191661 0.128675121 523 V L 0.443126307 0.088900743 760 G C 0.442544743 0.174174731 27 P T 0.442229152 0.271402709 694 G D 0.441607057 0.430247861 695 E D 0.440698297 0.174763691 96 M I 0.440309501 0.212758418 234 G V 0.44028737 0.19450919 385 E D 0.440128169 0.19408182 744 Y H 0.439198298 0.25211241 519 Q H 0.438343378 0.164581049 385 E [stop] 0.438258279 0.212771705 793 S R 0.438010456 0.160112082 726 A S 0.437983799 0.129329735 953 D Y 0.437888499 0.29124605 203 E [stop] 0.437866757 0.193004717 887 G V 0.437831028 0.150855683 189 G R 0.437816981 0.195105194 672 P L 0.437768207 0.1420574 906 Q R 0.437668081 0.257388395 887 G R 0.436446894 0.261046568 6 I T 0.436255483 0.311769796 751 M R 0.436212653 0.194544034 115 V A 0.436134597 0.191229151 490 R G 0.435740618 0.182925074 789 E G 0.435579914 0.162786893 603 — LE 0.43556049 0.202470667 442 R S 0.435504028 0.210966357 714 R I 0.435462316 0.200883142 8 K R 0.435212211 0.195908908 854 N D 0.43513717 0.067943636 335 E [stop] 0.434927464 0.21407853 915 G R 0.434895859 0.195491247 762 G C 0.434868342 0.215911162 3 I T 0.434607673 0.107252687 406 E [stop] 0.434574625 0.271888642 710 V A 0.434488312 0.161462791 594 E Q 0.434478655 0.199232108 601 L M 0.433295669 0.21298138 194 — DFY 0.433205 0.315807396 79 A S 0.433187114 0.14702693 913 NC FS 0.432811714 0.214195068 955 R S 0.432632415 0.15138175 793 — SKTYL (SEQ ID NO: 0.432421193 0.207758327 3683) 171 P H 0.432364213 0.194710101 560 N S 0.432346515 0.239882019 370 — GYK 0.432297106 0.219290605 321 P Q 0.432271564 0.211438092 979 LE[stop]GS-PG VSSKDLRA (SEQ ID 0.432126183 0.250028634 (SEQ ID NO: NO: 3684) 3668) 21 K E 0.431813708 0.20570077 348 C W 0.431395847 0.285738532 712 Q E 0.430794328 0.137430622 867 V A 0.430546539 0.112438125 902 H N 0.430482041 0.210989962 232 C R 0.430431738 0.130635142 164 E [stop] 0.43010378 0.307258004 348 C R 0.429790014 0.254295816 13 L R 0.429496589 0.209797858 11 R W 0.429311947 0.298268587 944 Q E 0.429084418 0.194128082 974 K E 0.428778767 0.120819051 935 L M 0.428357966 0..108223034 131 Q E 0.427961752 0.108783149 961 W R 0.427770336 0.153009954 508 F L 0.427277307 0.150834085 732 D Y 0.427260152 0.232782252 876 S G 0.427219565 0.1654176 35 M I 0.426965901 0.18021585 699 E [stop] 0.426936027 0.247620152 624 R G 0.426915666 0.161800086 687 — PTHIL (SEQ ID NO: 0.426399688 0.235010897 3685) 176 A G 0.425859136 0.154112817 256 K N 0.425760398 0.195398586 904 P A 0.425684716 0.273763449 859 Q K 0.425619083 0.166409301 222 G [stop] 0.425285813 0.299517445 20 K E 0.425128158 0.147645138 327 G C 0.425002655 0.239317573 530 L P 0.423859206 0.240275284 175 E Q 0.423850119 0.242087732 797 L P 0.423394833 0.254739368 351 K M 0.423313443 0.177944606 912 L M 0.423204978 0.27824291 188 F L 0.422539663 0.187750751 850 I M 0.422159968 0.218452121 391 K N 0.422162984 0.158915852 894 — S 0.42194087 0.23660887 758 S R 0.420859106 0.119214586 941 K N 0.420814047 0.266042931 381 L P 0.42076192 0.122089029 926 L V 0.42049552 0.169568285 873 S R 0.420222785 0.189220359 823 R G 0.420141589 0.140425724 703 T A 0.419927183 0.299947391 265 K N 0.419762272 0.205398427 904 P L 0.419717349 0.24717221 315 G A 0.419275038 0.167267502 346 M I 0.418933456 0.153077303 301 V A 0.418922077 0.253824177 545 I M 0.418607437 0.264461321 676 P T 0.41817469 0.167866208 516 F S 0.418152987 0.18301751 790 G V 0.417872524 0.178000118 890 G V 0.417424955 0.242331279 684 L P 0.41697175 0.237298169 369 A T 0.416965887 0.158164268 890 G R 0.416918523 0.30183511 515 A I 0.416763488 0.158965629 903 R G 0.416689964 0.149830948 898 K [stop] 0.416641263 0.154852179 632 L V 0.416523782 0.131108293 126 G D 0.41639346 0.171080754 151 H R 0.41621118 0.192083944 480 L P 0.4153828 0.153349872 569 M I 0.415261579 0.12705723 819 A S 0.414776737 0.173259385 212 E [stop] 0.414560972 0.214325617 104 P T 0.414121539 0.241680787 765 G A 0.413859942 0.202334164 862 — VK 0.413059952 0.195129021 210 P A 0.412638448 0.228860931 824 V A 0.412207035 0.173953175 736 N K 0.411883437 0.18103448 13 L H 0.411795935 0.405614507 844 L V 0.411372197 0.244473235 564 G C 0.411344604 0.228201596 694 G R 0.41123482 0.211796515 977 V L 0.411157664 0.380351062 142 E K 0.410509302 0.15102557 4 K E 0.410380978 0.274892917 890 G D 0.410337543 0.240602631 409 H D 0.410132391 0.22531365 563 S C 0.409998896 0.206123321 793 S N 0.409457982 0.067541166 705 Q H 0.409365382 0.15278139 515 A D 0.409252018 0.206051204 382 S R 0.408669778 0.157144259 97 S N 0.408564877 0.109922347 624 R I 0.40845718 0.228955853 568 P T 0.408066084 0.284742394 702 R S 0.408063786 0.129537189 796 Y N 0.40788333 0.311628718 897 K R 0.407876662 0.136002906 292 A V 0.407642755 0.163883385 741 L Q 0.407532982 0.11928093 315 G C 0.407147181 0.218556644 −1 S Y 0.407080752 0.324937034 945 T I 0.407011152 0.285905433 695 E [stop] 0.406081569 0.227028835 956 A S 0.405686952 0.185566124 752 L M 0.405575007 0.172103348 45 E [stop] 0.405531899 0.162357698 487 G C 0.405450681 0.290615306 310 Q R 0.405123752 0.12048192 791 L P 0.404916001 0.108993438 767 R I 0.404746394 0.223610078 538 G C 0.404409405 0.233295785 584 P A 0.403953066 0.108926305 552 A D 0.403929388 0.192995621 648 N D 0.403814843 0.290734901 973 W L 0.403521777 0.16358494 976 A S 0.403444209 0.261893297 180 L P 0.403389637 0.163854455 220 A S 0.402957864 0.279961071 894 — SLLKK (SEQ ID NO: 0.402797711 0.216370575 3686) 739 R I 0.402772732 0.234602886 548 E [stop] 0.402765683 0.262561545 764 Q K 0.402617217 0.220740512 723 A D 0.402461227 0.236080429 934 F L 0.402458138 0.384373835 42 E D 0.401939693 0.171540664 956 A G 0.401859954 0.23877341 771 A D 0.401428057 0.231350403 15 K M 0.401237871 0.256454456 298 A V 0.401000777 0.140487597 128 A P 0.400992369 0.173078759 511 Q H 0.400978135 0.171613013 26 G V 0.400800405 0.212307845 591 — QGREFI (SEQ ID 0.400574847 0.190655853 NO: 3687) 156 G S 0.400389686 0.306653761 728 N S 0.400298817 0.177178828 917 — ETHADE (SEQ ID 0.400170477 0.15562198 NO: 3688) 640 R G 0.399931978 0.200741 254 I M 0.39981124 0.209846066 644 L P 0.399481964 0.165702888 549 A S 0.399416255 0.189530269 528 L V 0.399354304 0.147818268 502 I V 0.399285899 0.256373682 79 A D 0.399080303 0.154917165 753 I M 0.399024046 0.268887392 588 G D 0.398941525 0.112261489 722 Y H 0.398538883 0.164012123 550 — G 0.398527591 0.353355602 133 C R 0.398285042 0.283233819 591 — QG 0.398079043 0.133460692 877 V L 0.398057665 0.212468549 958 V A 0.398007545 0.130004197 903 R I 0.39789959 0.321002606 118 G D 0.397657151 0.192339782 745 A S 0.397594938 0.285476509 914 C F 0.397278541 0.29475166 461 — SFV 0.39704755 0.20205322 637 — TFE 0.396824735 0.209304074 855 R M 0.396780958 0.191874811 142 E [stop] 0.396624103 0.229993954 108 D N 0.396298431 0.15939576 730 — ADDMVRN (SEQ ID 0.395727458 0.207712648 NO: 3689) 241 T I 0.395690613 0.131948289 641 R I 0.395315387 0.202249461 364 F L 0.395209211 0.112951976 739 R G 0.395162717 0.191317885 446 A S 0.39510798 0.254001902 593 R [stop] 0.395071199 0.196636879 168 L P 0.39502304 0.27101743 890 G C 0.394653545 0.224530018 677 — LS 0.394551417 0.187547463 47 L R 0.394492318 0.238759289 339 N S 0.394.482682 0.152047471 316 R G 0.394439897 0.159274636 206 H N 0.394299838 0.156799046 651 P A 0.394024946 0.151434436 441 R G 0.393551449 0.150649913 325 L P 0.393343386 0.140601419 589 K N 0.3926379 0.261890195 873 S G 0.392619693 0.143564629 414 G D 0.392615344 0.149137614 237 A G 0.392578525 0.167793454 479 E [stop] 0.392365621 0.272905538 752 L V 0.392234134 0.171880044 692 R I 0.391963575 0.221910688 683 S Y 0.39187962 0.197184801 568 P S 0.391506615 0.094807068 114 P I 0.391456539 0.163794182 341 V A 0.391246425 0.087691935 50 K R 0.39108021 0.159163965 698 K R 0.390885992 0.181654156 979 L- V[stop] 0.3907803 0.18994351 932 W G 0.390757599 0.185057669 519 Q R 0.390675235 0.117792262 140 K E 0.390615529 0.123713502 40 L P 0.390579865 0.194510846 978 — [stop] 0.390537744 0.255501032 509 S T 0.390466368 0.117704569 465 E [stop] 0.390424913 0.211758729 88 F S 0.390363974 0.156430305 429 E [stop] 0.390336598 0.135919503 783 — TAK 0.390178711 0.143499076 442 R M 0.390097432 0.262199628 453 T A 0.389911631 0.312187594 923 Q H 0.389855175 0.353446475 666 V A 0.389840585 0.169825945 499 E D 0.38958943 0.172940321 930 R G 0.389517964 0.2357312 847 — EGQITY (SEQ ID 0.389324278 0.122951036 NO: 3690) 846 V L 0.389120343 0.259313474 908 K N 0.38907418 0.225076472 975 P T 0.388901662 0.256059318 149 K N 0.38882454 0.171027465 691 L P 0.388805401 0.14397393 207 P A 0.387921412 0.102883658 11 — S 0.387747808 0.379461072 638 F L 0.387272475 0.168477543 558 V L 0.386662896 0.254612529 816 I V 0.386659025 0.185203822 680 F L 0.386638685 0.211225716 329 P T 0.386489681 0.220048383 576 D G 0.386151413 0.113653327 225 G V 0.386137184 0.239109613 22 A G 0.385839168 0.336984972 146 D E 0.385277721 0.095712474 507 G R 0.385233777 0.212044164 523 V I 0.385109283 0.152511446 501 S G 0.385073546 0.140125388 763 R L 0.38502172 0.191531655 705 Q E 0.384851421 0.17568818 82 H D 0.383907018 0.103874584 794 K N 0.383803253 0.195192527 979 LE[stop]GSPG VSSKDLR (SEQ ID 0.38375861 0.240184851 (SEQ ID NO: NO: 3691) 3668) 894 S R 0.383344078 0.273603195 639 E [stop] 0.383174826 0.193125393 655 I M 0.383102617 0.208514699 261 L V 0.382856978 0.19611714 480 L R 0.382841683 0.252187108 489 L V 0.38262991 0.16124555 134 Q E 0.382580711 0.180510987 650 — PA 0.382487274 0.372015728 630 P H 0.381699363 0.211396524 21 K R 0.381603442 0.1634713 677 — LSR 0.381372384 0.163400905 284 P T 0.381276843 0.171865261 783 T R 0.381262501 0.118770396 916 F V 0.380756944 0.281228145 450 A T 0.38074186 0.136570467 906 Q E 0.380700478 0.285392821 29 K [stop] 0.380574061 0.171976662 936 R I 0.38042421 0.204558309 754 F I 0.380277272 0.145574058 315 G S 0.380117687 0.143338421 89 Q [stop] 0.379768129 0.102222221 289 G C 0.379664161 0.235845043 750 A T 0.379378398 0.182932261 216 G C 0.379274317 0.176888646 303 W C 0.379215164 0.182222922 295 N K 0.379144284 0.378487654 919 H Y 0.379137691 0.321018649 726 A D 0.379067543 0.145080733 133 C S 0.378841599 0.162936296 497 E [stop] 0.378292682 0.202801468 444 E K 0.378042967 0.318660643 693 I M 0.378036899 0.225823359 587 F L 0.377947216 0.117981043 291 E D 0.377733323 0.142365006 85 W S 0.377648166 0.097279693 165 R M 0.377647305 0.161201002 569 M I 0.377387614 0.195898876 247 I I 0.37729282 0.165305688 513 — N 0.377106209 0.14731404 754 F L 0.376911731 0.164266559 21 K [stop] 0.376868031 0.199468055 268 A T 0.376839819 0.129211081 672 P T 0.376830532 0.204970386 735 R [stop] 0.376814295 0.09621637 147 K E 0.376789616 0.140417542 904 P R 0.37666328 0.185106225 712 Q H 0.376030218 0.227827888 2 E V 0.375325693 0.197955097 184 S I 0.375300851 0.252137747 163 H D 0.3751698 0.208290707 677 L P 0.375131489 0.090158552 44 L P 0.374906966 0.249472829 606 G V 0.374739683 0.285964981 937 S G 0.374669762 0.248499289 727 K N 0.374273348 0.164838535 734 D A 0.374244799 0.121134147 902 H Q 0.374087073 0.175219897 398 F L 0.373909011 0.239653674 845 K N 0.373742099 0.158752661 822 D N 0.373424135 0.138952336 136 L M 0.372880562 0.202180857 543 K E 0.372880222 0.146877967 244 Q H 0.372873077 0.184616643 403 L R 0.372697479 0.330913239 679 R I 0.372176403 0.370324076 738 A D 0.372074442 0.291834989 155 F L 0.371845015 0.114679195 174 P R 0.371603352 0.137168151 919 H N 0.371556993 0.327290993 944 Q H 0.37144256 0.338788753 164 E G 0.370935537 0.216755032 197 S G 0.370856052 0.178568608 840 N K 0.370814634 0.142530771 13 L M 0.370495333 0.29166367 488 D N 0.370055302 0.226946737 929 A P 0.370027168 0.168555798 580 L V 0.36995513 0.139984948 135 P A 0.369933138 0.10604161 342 D Y 0.369924443 0.189241086 959 ET AV 0.369879201 0.114167508 557 T A 0.369640872 0.087836911 6 I V 0.369460173 0.192497769 92 P T 0.368981275 0.236532466 292 A T 0.36879806 0.193425471 465 E D 0.36752489 0.224455423 189 — GQRALDFY (SEQ ID 0.368745456 0.227136846 NO: 3692) 805 T A 0.368671629 0.11272788 947 K E 0.368551642 0.227968732 148 G D 0.36788165 0.139635081 129 C W 0.367758112 0.199915902 129 C [stop] 0.367708546 0.192643557 98 R T 0.367673403 0.174398036 478 C W 0.367598979 0.111931907 228 L M 0.367328433 0.24869867 547 P H 0.367324308 0.220855574 105 K N 0.367245695 0.155463083 597 W R 0.367058721 0.142955163 328 F L 0.366955458 0.100787228 469 E [stop] 0.366917206 0.180496612 130 S T 0.366622403 0.127263853 283 Q E 0.366530641 0.247989672 958 V L 0.366470474 0.270699212 673 E Q 0.366346139 0.219545941 118 G C 0.366255984 0.265748809 848 G V 0.366195099 0.200861106 923 G L 0.366184575 0.233234243 357 K R 0.366148171 0.185792239 623 — RRTRQD (SEQ ID 0.365486053 0.26101804 85 W C 0.365346783 0.146084706 376 — ALLPY (SEQ ID NO: 0.365321474 0.191317647 3694) 356 E D 0.365050343 0.136074432 262 A S 0.365012551 0.204615446 765 G S 0.3649426 0.100657536 717 — GYSR (SEQ ID NO: 0.364903794 0.186125273 3695) 199 H V 0.364586783 0.168211628 796 Y H 0.364521403 0.145575579 237 A P 0.364453395 0.150681341 768 T A 0.36435574 0.18512185 513 N D 0.364305814 0.16260499 823 RV LS 0.364237044 0.11377221 656 G A 0.364010939 0.135958583 276 P I 0.363878534 0.201304545 214 I V 0.363876419 0.142178855 300 I V 0.363823907 0.234997169 769 F S 0.363687361 0.079831237 182 T R 0.363686071 0.201742372 677 L V 0.363578004 0.138045802 796 Y C 0.363566923 0.281557418 5 R S 0.363258223 0.211185531 298 A S 0.36320777 0.211187305 594 E [stop] 0.36278807 0.205352129 105 K R 0.362205009 0.140104618 907 E Q 0.362024887 0.226228418 509 S G 0.361807445 0.13953396 110 R I 0.361752083 0.138681372 406 E Q 0.361750488 0.303638253 470 A V 0.361349462 0.10686226 4 K [stop] 0.36129388 0.179352157 362 K E 0.361196668 0.232368389 713 R G 0.3607467 0.181817788 857 K N 0.360715256 0.172046815 120 E D 0.36030686 0.214810208 277 K E 0.36002957 0.210892547 477 RCELK (SEQ ID SFSSH (SEQ ID NO: 0.360015336 0.177473578 NO: 3698) 3699) 532 I T 0.359759307 0.145072322 774 Q K 0.359747336 0.182131652 439 E D 0.359587685 0.134619305 198 I T 0.359370526 0.173615874 156 G C 0.359055571 0.173590319 399 G C 0.358922413 0.255017848 59 S I 0.358703019 0.109042363 93 V M 0.358615623 0.161948363 674 G [stop] 0.358503233 0.220631194 539 K N 0.358074633 0.087009621 709 E D 0.357944736 0.136689683 120 E G 0.357933511 0.168382586 494 F L 0.357874746 0.139367085 272 G V 0.357428523 0.207170798 527 N I 0.357320226 0.086164887 236 V A 0.357249373 0.125737046 974 K N 0.357242055 0.190403244 10 RR PG 0.356712463 0.324298272 39 D Y 0.356585187 0.235756832 579 N S 0.3558347 0.181516226 214 I M 0.355779849 0.142887254 843 E [stop] 0.355689249 0.225441771 526 — LNLY (SEQ ID NO: 0.355597159 0.179351732 3700) 667 I M 0.355548811 0.239632986 559 I V 0.355478406 0.171281999 706 A S 0.355431605 0.116949175 11 RR TS 0.35536352 0.272262643 865 L Q 0.355287262 0.164676142 946 N K 0.355277474 0.180093688 689 HI PV 0.355052108 0.144577201 898 K N 0.354894826 0.200062158 950 — GN 0.354845909 0.167057981 332 P T 0.354796362 0.20270742 323 Q E 0.354759964 0.249399571 42 E A 0.354721226 0.213005644 22 A T 0.354629728 0.083320918 948 T S 0.354488334 0.198422577 16 D E 0.354450775 0.187189495 170 S Y 0.354344814 0.160709939 862 — VKDLS (SEQ ID NO: 0.354059938 0.179170942 3701) 249 E [stop] 0.354016591 0.294486267 531 I M 0.353941253 0.095481374 266 D H 0.35392753 0.237329699 859 Q E 0.353923377 0.126451964 113 I V 0.353631334 0.187941798 136 L P 0.353572714 0.240617705 503 L M 0.353400839 0.174768283 51 P R 0.353321532 0.126698252 179 E D 0.353270131 0.108592116 31 L V 0.353260601 0.168619621 502 I F 0.353258477 0.139633145 378 L M 0.353221613 0.189998728 890 G A 0.353138339 0.149947604 913 N K 0.353092797 0.294888192 956 A D 0.352997131 0.204713576 158 C W 0.352758393 0.130405614 157 — RCNV (SEQ ID NO: 0.352566351 0.116984328 3702) 771 A G 0.352390901 0.141133059 227 A G 0.352335693 0.141777326 202 RE G- 0.352321171 0.210660545 99 V F 0.352314021 0.162936095 643 V E 0.352268894 0.209333581 41 R I 0.352205261 0.321737078 387 R P 0.352184692 0.159814147 539 K E 0.351957196 0.146275596 478 C F 0.351788403 0.313141443 942 K E 0.351775756 0.256493816 36 M I 0.351715805 0.097577134 644 L V 0.351676716 0.163471035 78 K E 0.35167205 0.128519193 272 G C 0.351365895 0.208785029 157 — RCNVSEHE (SEQ ID 0.351115058 0.126463217 NO: 3703) 883 S R 0.351093302 0.143213807 917 E V 0.350763439 0.206641731 843 E 0 0.350569244 0.142523946 870 D Y 0.350431061 0.194706521 393 F V 0.35027948 0.168738586 162 E K 0.350236681 0.12523983 119 N D 0.350147467 0.235898677 306 L M 0.349889759 0.165537841 110 R T 0.349523294 0.289863999 976 A D 0.34941868 0.2410.42383 914 C W 0.349231308 0.169568161 115 V M 0.349160578 0.17839763 863 K N 0.348978081 0.175915912 830 K R 0.348789882 0.11782242 564 G S 0.348654331 0.240781896 647 S I 0.348570495 0.163208612 617 E D 0.348384104 0.103608149 262 A T 0.348231917 0.222328473 713 R I 0.348163293 0.202182526 893 L P 0.348133135 0.24849422 202 R G 0.347997162 0.177282082 806 S Y 0.347673828 0.200543155 391 K R 0.347608788 0.122435715 683 S C 0.34755615 0.102168244 446 A I 0.347296208 0.236243043 282 P A 0.347073665 0.253113968 580 L P 0.347062657 0.078573865 895 L P 0.347059979 0.152424473 929 A I 0.34702013 0.306789031 108 D Y 0.347014656 0.291577591 258 E [stop] 0.34694757 0.281979872 673 E A 0.346691172 0.265253287 950 G D 0.346646349 0.128298199 792 P T 0.346487957 0.236073016 673 E [stop] 0.346388527 0.198074161 150 P R 0.34632855 0.278480507 456 L P 0.345951509 0.161500864 790 G R 0.345911786 0.179210019 647 S T 0.345819661 0.158521168 542 F S 0.3.45619595 0.191970857 841 G D 0.345447865 0.129392183 57 P A 0.345371652 0.147875225 578 P R 0.345346371 0.12075926 793 S I 0.3.45235059 0.262377638 453 T S 0.345118763 0.097101409 651 P R 0.345088622 0.208316961 556 Y [stop] 0.345070339 0.114662396 86 E [stop] 0.3.44943839 0.21976554 646 S G 0.344888595 0.154435246 592 G C 0.34478874 0.240350052 49 K N 0.344659946 0.130706516 586 A D 0.344294219 0.15117877 166 L V 0.34415435 0.139737754 726 A P 0.344144415 0.164178243 666 V L 0.344130904 0.155760915 749 D H 0.344052929 0.242192495 486 Y C 0.34395063 0.130965705 134 Q K 0.343594633 0.210709609 91 D H 0.34352508 0.153686099 40 LR PV 0.343506493 0.155292328 12 R T 0.343490891 0.187270573 653 N D 0.343487264 0.148663517 52 E Q 0.343438912 0.247941408 8 K Q 0.343298615 0.279455517 555 R L 0.343270194 0.098281937 294 N D 0.343264324 0.126839815 553 N D 0.342736197 0.153294035 893 L M 0.342736077 0.179172833 951 N K 0.342592943 0.278844401 51 P T 0.342576973 0.1929364 649 I T 0.342534817 0.270208479 175 E D 0.342455704 0.202360388 823 R S 0.341965728 0.273152096 219 C R 0.341954249 0.136482174 283 Q R 0.341949927 0.224313066 444 E [stop] 0.341881438 0.217688103 649 I V 0.341655494 0.148589673 854 N K 0.341614877 0.1579.48422 514 C S 0.34160113 0.231141571 623 — RRTR (SEQ ID NO: 0.341527608 0.187073234 3704) 585 L M 0.341496703 0.21431877 211 — LE 0.341207432 0.169230112 544 K E 0.341142267 0.208342511 478 C R 0.341091687 0.148433288 858 R G 0.340977066 0.206052559 172 H D 0.340873936 0.298188428 16 D A 0.340771918 0.308121625 525 K N 0.340626838 0.147516442 532 I V 0.340576058 0.099088927 520 K [stop] 0.34056167 0.228510512 743 Y [stop] 0.340397436 0.102396798 344 W C 0.340364668 0.176812201 220 A G 0.340276978 0.133945921 186 G V 0.340265085 0.116877863 694 G C 0.340225482 0.309935909 411 E Q 0.340144727 0.282548314 406 E G 0.340120492 0.140875629 573 F L 0.340030507 0.166015227 458 A G 0.339794018 0.171435317 675 C [stop] 0.339687357 0.208292109 576 D Y 0.339621402 0.21774439 787 A S 0.339526186 0.318305548 537 G C 0.339454064 0.174110887 185 — LG 0.339451721 0.186103153 844 L P 0.339318044 0.191881119 712 Q K 0.339288003 0.193891353 591 Q R 0.339223049 0.160616368 169 L P 0.339210958 0.127439702 923 — QAALN (SEQ ID 0.339143383 0.169170821 NO: 3705) 623 R S 0.339131953 0.245088648 589 K Q 0.33901987 0.177422866 522 G V 0.338985606 0.226282565 204 S T 0.338673547 0.170845305 698 K E 0.338580473 0.129708045 497 E V 0.338306724 0.13489235 23 G S 0.338162596 0.15304761 29 K R 0.337989172 0.147861886 716 G V 0.337974681 0.202399788 703 T S 0.337889214 0.141977828 979 LE[stop]GSPG VSSKDLE (SEQ ID 0.337814175 0.168342402 (SEQ ID NO: NO: 3706) 3668) 240 L M 0.3377179 0.151631422 950 G C 0.337265205 0.234973706 7 N S 0.337036852 0.185037778 64 A P 0.336967696 0.255179815 795 T S 0.336837648 0.117371137 480 L Q 0.336803159 0.213915334 600 L V 0.336801383 0.230766925 175 E [stop] 0.336712437 0.187755487 63 R S 0.336640982 0.183725757 394 A P 0.336388779 0.125201204 52 E [stop] 0.336207682 0.211986135 299 Q E 0.336024324 0.156699489 183 YS WM 0.335855997 0.179538112 194 D Y 0.335755348 0.131644969 213 Q R 0.335726769 0.209853061 802 A D 0.33571172 0.168573673 163 H N 0.33571123 0.197315666 943 Y C 0.335604909 0.172843558 118 G S 0.3355.14316 0.125891126 758 S G 0.335513561 0.149050456 941 K [stop] 0.335374859 0.192348189 279 — TLPPQPH (SEQ ID 0.335305655 0.144688363 NO: 3707) 632 LF PV 0.335263893 0.113883053 894 — SLLKKR (SEQ ID 0.335263893 0.141289109 NO: 3708) 943 V [stop] 0.335115123 0.291608146 38 P R 0.33481965 0.113021039 616 I F 0.334790976 0.107803908 134 Q H 0.334549336 0.158461695 186 G C 0.334321874 0.156717674 184 S G 0.334296555 0.223929833 765 G C 0.33423513 0.213904011 687 P T 0.334191461 0.22545553 803 — QYT 0.33418367 0.096860089 374 Q R 0.334175524 0.104826318 455 W C 0.334165051 0.186741008 552 — ANRFY (SEQ ID NO: 0.333923423 0.2586.49392 3709) 407 K R 0.333913165 0.142719617 175 E K 0.333834455 0.196225639 610 — LANGR (SEQ ID NO: 0.333428825 0.102899397 3710) 230 — DACM (SEQ ID NO: 0.333428825 0.108521075 3711) 848 G S 0.333406808 0.165245749 630 P R 0.333389309 0.182782946 442 R G 0.333281333 0.186150848 836 M T 0.33320739 0.215623837 222 G V 0.333139545 0.173506426 21 K I 0.333022379 0.190202016 696 S I 0.332955668 0.138037632 635 A T 0.332902532 0.130552446 551 E G 0.332833114 0.158314375 780 D Y 0.332787267 0.203141483 47 L M 0.332771785 0.228474741 347 V L 0.332766547 0.164853137 841 G C 0.332584425 0.2483922 593 R I 0.332546881 0.22140312 749 D Y 0.332359902 0.199451757 27 P S 0.332358372 0.306966339 276 P H 0.332221583 0.26420075 293 Y [stop] 0.332046234 0.133526657 3 I N 0.332004357 0.072687293 642 — EVLD (SEQ ID NO: 0.331972419 0.22538863 3712) 620 L P 0.331807594 0.15763111 456 L V 0.331754102 0.143226803 130 S G 0.331571239 0.167684126 629 E K 0.33154282 0.153428302 950 G V 0.331464709 0.229681218 328 F Y 0.331454046 0.090600532 303 W S 0.331070804 0.245928403 421 W C 0.330779828 0.216037825 351 K R 0.330630005 0.142537112 498 A T 0.33049042 0.166213318 937 S I 0.330380882 0.231058955 592 GR DN 0.329593548 0.300041765 127 F I 0.329561201 0.268089932 837 T S 0.329510402 0.099725089 704 I I 0.329114566 0.113551049 387 R L 0.328928103 0.199189713 171 P R 0.328685191 0.279786527 767 R T 0.328611454 0.173820273 597 W L 0.328585458 0.282536549 955 R G 0.328533511 0.252801289 629 E [stop] 0.328472442 0.226070443 699 E G 0.328340286 0.161755276 564 G A 0.328244232 0.11512512 129 C F 0.327975914 0.184885596 26 D S 0.327861024 0.174859434 199 H N 0.327823226 0.25147122 701 Q R 0.327746296 0.151982714 186 G D 0.327613843 0.101552272 422 E D 0.327579534 0.227939955 924 A T 0.327501843 0.29494568 176 A P 0.32741005 0.239900376 499 E K 0.327284744 0.159757942 546 K R 0.327156617 0.166513946 556 Y H 0.327151432 0.118520339 548 — EAF 0.326965289 0.171181066 901 S I 0.326880206 0.3201.48616 14 V I 0.326870011 0.276842054 814 F L 0.32685269 0.084563864 157 — RCNVSE (SEQ ID 0.326801479 0.200654893 NO: 3713) 250 H R 0.326584294 0.078102923 730 A V 0.326443401 0.110931779 497 E Q 0.326193187 0.212891542 536 K R 0.326129704 0.20597101 906 Q P 0.326073598 0.193779388 243 Y D 0.326001836 0.130392708 798 S F 0.325769587 0.320454172 882 S G 0.325732755 0.141569252 759 R G 0.325319087 0.080028833 576 D V 0.325192282 0.239519469 309 W [stop] 0.325098891 0.096106342 554 R I 0.325075441 0.185726803 483 Q H 0.324598695 0.153049426 979 -E VSSKDQ (SEQ ID 0.324398559 0.118712651 NO: 3714) 834 G C 0.324348652 0.175539945 719 S Y 0.324298439 0.22105488 842 K R 0.324267597 0.102772814 97 S T 0.324252325 0.240123255 172 H N 0.32.4047776 0.168532939 692 R G 0.324024313 0.134914995 39 D V 0.324012084 0.186802864 776 T I 0.323918216 0.153171775 652 M I 0.323898442 0.13705991 611 A V 0.323836429 0.18975125 658 D G 0.323834837 0.116577804 158 C [stop] 0.323773158 0.093674966 887 G A 0.32369757 0.19151617 337 Q H 0.323607141 0.165283008 319 A D 0.323458799 0.152084781 215 — GGNSCA (SEQ ID 0.323334457 0.165215546 NO: 3715) 351 K N 0.323273003 0.138737748 878 — I 0.323133111 0.265099492 597 W C 0.323039345 0.210227048 85 W G 0.3230112 0.140970302 830 K E 0.322976082 0.171606667 193 — LD 0.322600674 0.167338288 350 V A 0.32248331 0.252994511 786 L Q 0.32241581 0.22201146 4 K M 0.32231147 0.124043743 781 W R 0.322196176 0.263818038 182 T I 0.322044203 0.109310181 888 R G 0.322001059 0.172130189 388 K N 0.321769292 0.13958088 504 D Y 0.321517406 0.182186572 260 R I 0.321461619 0.146534668 695 E Q 0.321451268 0.199405121 960 T A 0.321351275 0.243570837 496 I F 0.321275456 0.162860461 454 D H 0.321034191 0.123925099 859 Q H 0.321009248 0.15665955 432 S I 0.32093586 0.219919612 120 E Q 0.320905282 0.134126668 359 E [stop] 0.320840565 0.172779106 474 E [stop] 0.320753733 0.198938474 609 K R 0.320654761 0.097190768 654 L P 0.320340402 0.21351518 344 W G 0.32013599 0.133467654 629 E D 0.319764058 0.097801219 631 A D 0.319695703 0.120854121 124 S Y 0.319588026 0.148095027 244 Q R 0.319581236 0.174412151 338 A D 0.319500211 0.171228389 634 V L 0.3194918 0.113193905 91 D N 0.319468455 0.231799127 740 D E 0.319148668 0.093677265 942 K R 0.319440348 0.184998826 146 D Y 0.319268754 0.209601725 513 N K 0.319264079 0.180017602 366 Q H 0.318971922 0.184226775 477 R G 0.318963003 0.179227033 947 K R 0.318930494 0.25585521 478 C S 0.318576968 0.151506435 443 S G 0.318453544 0.181417518 766 K E 0.318255467 0.119279294 557 T S 0.318254881 0.136960287 39 D E 0.318241109 0.177504749 586 A S 0.318046156 0.197164692 270 A P 0.317952258 0.133471459 707 A S 0.317797903 0.176472631 173 K N 0.317699885 0.158843579 676 P R 0.317616441 0.273323665 409 H N 0.31739526 0.238962249 878 N D 0.317341485 0.123856244 967 K E 0.317328223 0.198885809 405 L M 0.317316848 0.232382071 759 R I 0.317284234 0.210047842 505 I M 0.317274558 0.129635964 612 N D 0.317252502 0.181380961 862 V A 0.317158438 0.090072044 295 -N LS 0.317076665 0.155046903 165 R G 0.317047785 0.17842685 760 G D 0.316786277 0.162885521 244 Q K 0.316600083 0.246636704 238 S Y 0.316596499 0.171458712 475 F L 0.316549309 0.192939087 829 K N 0.316494901 0.154808851 28 M I 0.31630177 0.188404934 186 G A 0.316262682 0.1767869 679 R G 0.316180477 0.112760057 925 A G 0.315901657 0.192750307 892 A P 0.315901657 0.129374073 642 E A 0.315758891 0.205380131 629 E G 0.315702888 0.119743865 642 E G 0.315673565 0.11044042 104 P R 0.315607101 0.202791238 807 K E 0.315573228 0.117464708 599 D E 0.315416693 0.115740153 94 G A 0.315344942 0.125574217 509 S R 0.315237336 0.198196247 715 A S 0.314795788 0.184022977 639 E G 0.314490675 0.131536259 485 W R 0.314444162 0.077460473 529 Y [stop] 0.314338149 0.096977512 773 R M 0.314128132 0.191934874 227 A D 0.313893012 0.086820124 865 L V 0.313870986 0.093939035 25 T S 0.313828907 0.165926738 206 H R 0.313540953 0.153060153 33 V I 0.313378588 0.092743144 736 N S 0.313292021 0.139875641 613 G A 0.313219371 0.139952239 472 K R 0.313201874 0.163543589 149 — KPH 0.313073613 0.111009375 966 R I 0.313069041 0.220268045 847 E [stop] 0.312986862 0.248850102 892 A V 0.312917635 0.236911004 322 L P 0.312907638 0.167614176 947 K N 0.312809501 0.23804854 820 D Y 0.312669916 0.196444965 627 Q E 0.312477809 0.180929549 20 K T 0.312450252 0.306509245 914 C G 0.312434698 0.246328459 793 S G 0.312385644 0.182436917 411 P D 0.312132984 0.213313342 901 S R 0.311953255 0.163461395 393 F L 0.311946018 0.192991506 757 L P 0.311927617 0.117197609 702 R G 0.311688104 0.266620819 589 K R 0.311588343 0.136320933 717 G R 0.311565735 0.080863714 286 T S 0.311321567 0.240949263 150 P T 0.311291496 0.13427262 578 P A 0.311263999 0.106013626 41 R G 0.311016733 0.286865829 781 W S 0.310870839 0.281958829 382 S I 0.310857774 0.22558917 723 A T 0.310856537 0.118165477 451 A G 0.310527551 0.159640493 568 P L 0.310447286 0.186724922 216 G S 0.310362762 0.143843218 216 G R 0.310272111 0.119909677 89 Q R 0.310167676 0.139047602 433 K R 0.310161393 0.097615554 21 KA NC 0.310061242 0.098851828 141 L P 0.309573602 0.118441502 425 D Y 0.309531408 0.253195982 579 N D 0.309484128 0.137585893 825 L V 0.309431153 0.160157183 464 I M 0.309049855 0.208541437 710 V L 0.309047105 0.126001585 671 D H 0.309035221 0.209514286 735 R P 0.309028904 0.132025621 819 A G 0.308778739 0.188847749 2 E G 0.308512084 0.159248809 109 Q H 0.308384304 0.180580793 66 L V 0.308337109 0.160085063 93 V L 0.308334538 0.186355769 621 Y [stop] 0.308307714 0.182192979 0 M L 0.308276685 0.236934633 857 K E 0.308118374 0.128063493 264 L I 0.308089176 0.231951197 646 S T 0.307934288 0.163215891 461 S T 0.307923977 0.13026743 937 S N 0.307902696 0.280386833 774 Q L 0.30782826 0.179585187 427 K N 0.307771318 0.212433986 422 E G 0.307743696 0.21393123 107 I L 0.307707331 0.205313283 776 T A 0.307705621 0.113209696 306 L V 0.307515106 0.116397313 651 P I 0.307457933 0.189846398 155 F Y 0.307385155 0.165676404 229 S I 0.307373154 0.086318269 517 I V 0.307363772 0.108604289 334 V A 0.306982037 0.139604112 614 R K 0.306921623 0.187827913 824 V L 0.306719384 0.210851946 723 A V 0.306692766 0.140247988 711 E G 0.306675894 0.224133351 499 E Q 0.306671973 0.224590082 104 P S 0.306640385 0.162249455 3 I L 0.306608196 0.194776786 702 R K 0.306541295 0.149431609 954 K E 0.306525004 0.187285491 842 — KEL 0.306410776 0.206532128 466 G C 0.30635382 0.179163452 979 — VSSKD (SEQ ID NO: 0.306277048 0.179502088 3673) 830 K [stop] 0.306086752 0.154175951 243 Y F 0.306073033 0.15669665 88 F L 0.305867737 0.156711191 149 K E 0.305762803 0.092392237 102 P H 0.305663323 0.198476248 554 — RFYI (SEQ ID NO: 0.305511625 0.122801047 3716) 720 — R 0.305347434 0.161540535 128 A G 0.305254739 0.159245241 122 L P 0.305222365 0.154910099 792 P S 0.305214901 0.160903917 312 L P 0.305192803 0.183880511 299 Q [stop] 0.305119863 0.096364942 668 A T 0.305069729 0.135204642 639 E Q 0.304680843 0.266883075 812 C [stop] 0.304671385 0.223383408 856 — YK 0.304562199 0.117931145 959 — ETWQSFY (SEQ ID 0.304562199 0.204359044 NO: 3717) 640 R [stop] 0.304365031 0.131009317 968 KL S[stop] 0.304328899 0.221090558 24 K N 0.304215048 0.239991354 858 R T 0.304052714 0.1448623 530 L M 0.303970715 0.250168829 269 S R 0.303928294 0.209763505 251 Q E 0.303459913 0.190095434 340 E Q 0.30343193 0.10804688 623 — R 0.303430789 0.233394445 880 D Y 0.30324465 0.244720194 223 P A 0.303031527 0.177373299 899 R T 0.302967154 0.112177355 60 N D 0.30295183 0.177064719 966 R S 0.302926375 0.099801177 687 P A 0.302859855 0.188291569 821 Y C 0.302780706 0.154234626 628 D Y 0.302709978 0.176578494 952 — TDKRAFVE (SEQ ID 0.302629733 0.089246659 NO: 3718) 540 L V 0.302623885 0.094608809 855 R T 0.302608606 0.19469877 59 S I 0.302606901 0.165051866 272 G D 0.302541592 0.185286895 284 P H 0.302498547 0.213421981 342 — TS 0.302413033 0.240972915 43 R W 0.302283296 0.149981215 760 G A 0.302207311 0.130376601 766 K N 0.302181165 0.136382512 962 Q R 0.302114892 0.192863031 656 G S 0.301941181 0.160658808 526 L P 0.301907253 0.200130867 181 V L 0.301627326 0.141701986 602 S G 0.301374384 0.168690577 2 E K 0.301361669 0.293245611 46 N S 0.301357514 0.121526311 71 T S 0.301285774 0.182156883 887 G D 0.301271887 0.117733719 121 R S 0.301231571 0.167844846 108 D V 0.301094262 0.261979025 979 LE[stop]GS-PGi VSSKDLQA (SEQ ID 0.301043 0.222937332 (SEQ ID NO: NO: 3671)[stop] 3674) 73 Y [stop] 0.300976299 0.109164204 645 D H 0.300832783 0.189820783 972 — VWK 0.300386808 0.146545616 127 F S 0.300342022 0.146847301 571 V A 0.300337937 0.156010497 386 D N 0.300273532 0.259491112 381 L M 0.300116697 0.157006178 493 P A 0.299995588 0.227049942 199 H R 0.299830107 0.074234175 642 E [stop] 0.299768631 0.20842894 352 K [stop] 0.299555207 0.106916877 314 I V 0.299339024 0.237860572 696 S T 0.299269551 0.19370537 554 R G 0.299260223 0.263070996 413 W S 0.298889603 0.120871006 973 W [stop] 0.298886432 0.173734887 1 Q [stop] 0.298848883 0.253324527 59 S G 0.298416382 0.178538741 717 G [stop] 0.298317755 0.217662606 348 C S 0.298274049 0.13599769 707 A G 0.298173789 0.189062395 478 CE AQ 0.298056287 0.28697996 915 G A 0.298020743 0.21282862 969 L M 0.297993119 0.288243926 953 D V 0.297929214 0.145206254 485 W G 0.297911414 0.242181721 676 P A 0.297863971 0.0896.40148 4 K T 0.297828559 0.161108285 631 A G 0.297777083 0.103836414 250 H P 0.29766948 0.081415922 11 — R 0.29755173 0.242218951 274 A T 0.297540582 0.172279995 918 T K 0.297381988 0.249593921 43 R L 0.297375059 0.247052829 51 P A 0.29736536 0.241677851 64 A T 0.297190007 0.136022098 617 E Q 0.297156994 0.256789508 468 — K 0.297121715 0.218726347 705 Q [stop] 0.297097391 0.129530594 538 G D 0.297030166 0.143641253 697 Y [stop] 0.29694611 0.165401562 30 T N 0.296922856 0.20113666 374 Q E 0.296916876 0.294201034 429 E G 0.296692622 0.12956891 617 E G 0.296673186 0.100617287 174 P L 0.296325925 0.125090192 476 C W 0.296243077 0.108583652 536 K [stop] 0.296174047 0.204485045 340 E [stop] 0.296106359 0.228363644 263 N S 0.295761788 0.153417105 292 A D 0.295588873 0.132003236 524 K E 0.295588726 0.123024834 252 K E 0.295509892 0.130412924 360 D H 0.295426779 0.169820671 771 A T 0.295409018 0.21146028 960 T S 0.295303172 0.200733126 345 D Y 0.295298688 0.153403354 469 E G 0.295269456 0.193145904 495 A T 0.295248074 0.179130836 929 A G 0.295233981 0.250007265 435 I T 0.2952095 0.10707736 586 A T 0.295123473 0.125804414 627 Q R 0.295089748 0.147312376 17 S I 0.295022842 0.203345294 96 M V 0.29492941 0.118289949 83 V M 0.294841632 0.151911965 721 K [stop] 0.294783263 0.121804362 550 F S 0.294772324 0.160417343 538 G A 0.29474804 0.174345187 462 F L 0.294742725 0.14185505 822 D H 0.294658575 0.162957386 213 QI PV 0.294575907 0.193654125 658 D N 0.294502464 0.107952026 309 W S 0.294338009 0.284836107 835 W C 0.294317109 0.120763755 607 S V 0.294194742 0.192145848 853 Y [stop] 0.294188525 0.116100881 895 L M 0.294152124 0.189733578 298 AQ DR 0.294067945 0.080730567 221 S T 0.293988985 0.161830985 854 — NRYKRQ (SEQ ID 0.29389502 0.164228467 NO: 3719) 184 — SLG 0.29389502 0.133943716 24 K E 0.293893146 0.087429384 903 R T 0.293855808 0.156130706 649 I M 0.293844709 0.213121389 646 S N 0.293718938 0.053702828 751 M T 0.293692865 0.188828745 138 V A 0.293692865 0.172441917 421 W R 0.293643119 0.202965718 885 T A 0.293639992 0.136222429 372 K N 0.293601801 0.159631501 899 R W 0.293409271 0.197663789 323 Q R 0.293396269 0.187618952 787 A V 0.293181255 0.111256021 97 S G 0.29311892 0.120983434 523 V A 0.293107836 0.144403198 606 GS -A 0.293095145 0.176419666 647 S G 0.293070849 0.180316262 401 L M 0.293059235 0.238931791 706 A T 0.293004089 0.157196701 167 I M 0.292976512 0.174801994 239 F Y 0.292846447 0.244049066 532 I M 0.292790974 0.132047771 362 K N 0.292779584 0.196868197 531 I F 0.292690193 0.245999103 551 E D 0.292676692 0.177028816 366 Q R 0.292637285 0.233099785 45 E K 0.292602703 0.135241306 170 S P 0.292187757 0.117055288 522 — GVKKLNLY (SEQ ID 0.292477218 0.205588046 NO: 3720) 184 S T 0.292461578 0.171099938 256 K R 0.292459664 0.134546625 898 K R 0.292371281 0.233917307 687 — PTHILR (SEQ ID NO: 0.292237604 0.252992689 3721) 499 E [stop] 0.292180944 0.205912614 439 E [stop] 0.291789527 0.178224776 286 T I 0.291597253 0.134630039 326 K R 0.291167908 0.130858044 309 W C 0.291117426 0.126634127 141 L V 0.291053469 0.125358393 599 D H 0.290990101 0.194898673 891 E D 0.290888227 0.199229012 663 I T 0.290884576 0.159824412 86 E G 0.290735509 0.164271816 950 — GNTDKRA (SEQ ID 0.290646329 0.08439848 NO: 3722) 910 V A 0.290614659 0.192165123 130 S R 0.290579337 0.126556505 286 T A 0.290569747 0.161258253 412 D Y 0.290563856 0.192946257 390 G C 0.290531408 0.226107283 96 M T 0.290483084 0.117441458 796 Y F 0.290480726 0.145066767 617 E [stop] 0.290459043 0.254049857 520 K Q 0.290432231 0.149193863 238 S C 0.29036146 0.125809391 510 K N 0.290307315 0.121616244 751 M I 0.290086322 0.117481113 764 Q E 0.290043861 0.213865459 239 F L 0.290032145 0.120563078 750 A S 0.290021488 0.169783417 509 S N 0.290010303 0.173158694 791 L V 0.28993006 0.240441646 976 A P 0.289917569 0.129909297 970 K E 0.289792346 0.088055606 370 G S 0.289754414 0.116500268 229 S I 0.289718863 0.192569781 126 G S 0.289695476 0.136718855 39 D H 0.28966543 0.205820796 541 R W 0.289647451 0.149474595 963 S R 0.289642486 0.119359764 614 R G 0.289631701 0.096593744 903 R K 0.289598509 0.276955136 700 K E 0.289582689 0.146563937 176 A T 0.289565984 0.071489526 714 R G 0.289551118 0.131217053 849 Q E 0.289450204 0.14256548 861 V L 0.289424991 0.184715842 227 A S 0.289407395 0.147147965 337 Q E 0.289400311 0.154536453 282 P Q 0.289371748 0.241776764 147 — KGKPH (SEQ ID NO: 0.289327222 0.167067239 3723) 215 — GGNSCASG (SEQ ID 0.28926976 0.113347286 NO: 3724) 615 — Q 0.288918789 0.138819471 148 — GKPHTNY (SEQ ID 0.288918789 0.145077971 NO: 3725) 70 L V 0.288897546 0.141249384 131 Q H 0.28889109 0.089984222 417 Y [stop] 0.288830461 0.139069155 917 P Q 0.288684907 0.209421131 681 K R 0.288657171 0.188212382 824 — VLE 0.288568311 0.142383803 757 L M 0.288547614 0.138199941 683 S P 0.288449161 0.100064584 879 N D 0.288359669 0.112916417 87 EF AV 0.28833835 0.157423397 623 R M 0.288312668 0.180378091 360 D G 0.288240177 0.1450193 469 E D 0.288213424 0.169330277 488 D H 0.288056714 0.224399768 832 A D 0.28797086 0.133987122 331 F L 0.287898632 0.125465761 880 D N 0.287796432 0.265861692 813 G V 0.28764847 0.18793522 125 S R 0.287612867 0.078156909 315 G V 0.287582891 0.216366011 862 V L 0.28755723 0.122530143 376 A D 0.287488687 0.149852687 717 G A 0.287475979 0.138371481 871 R G 0.287423469 0.12544588 779 P [stop] 0.287388451 0.214465092 659 R Q 0.287382153 0.188389105 688 T S 0.2872606 0.18090055 450 A G 0.287222025 0.226851871 608 L P 0.287206606 0.153956956 74 T A 0.28708898 0.151009591 101 Q H 0.287075864 0.127870371 168 L M 0.287051161 0.164606192 522 G A 0.286889556 0.191392288 158 — CN 0.286856801 0.104191954 822 D Y 0.286792384 0.216414998 31 LL PV 0.286704233 0.167404084 753 — IFENLS (SEQ ID NO: 0.286664247 0.204891377 3726) 894 — SLLK (SEQ ID NO: 0.286588033 0.088926565 3727) 443 S R 0.286575868 0.16053834 813 G S 0.286517663 0.166687094 545 I T 0.28643634 0.175437623 43 R G 0.286322337 0.211707784 671 D G 0.28629192 0.163952723 501 S T 0.286282753 0.120251174 729 L M 0.286200559 0.141100837 264 L F 0.28603772 0.148836446 613 G P 0.285821749 0.213295055 806 S P 0.285754508 0.139734573 251 Q R 0.285704309 0.129794167 503 L P 0.285623626 0.150765257 544 K N 0.285528499 0.105740594 685 G S 0.285482686 0.116956671 66 L P 0.285241304 0.178235911 348 C [stop] 0.285167016 0.232120541 615 V L 0.285139566 0.138644746 34 R K 0.285068253 0.155629412 606 G D 0.284708065 0.131937418 564 G R 0.284584869 0.153328649 767 R G 0.284520477 0.167110905 459 K N 0.284319069 0.144116629 100 A G 0.284064196 0.232698011 182 T S 0.284017418 0.165066704 552 A P 0.28399207 0.192922882 874 E [stop] 0.283924403 0.212096559 656 G V 0.283837412 0.096364514 527 N D 0.283828964 0.095606466 560 N D 0.283827293 0.131100485 518 W [stop] 0.283768829 0.144873432 900 F Y 0.283754684 0.18210141 485 W C 0.283722783 0.101623525 528 L M 0.283582823 0.241404553 463 V L 0.283409253 0.174572622 938 Q R 0.283399277 0.159588016 809 C R 0.2832933 0.140866937 765 G V 0.283226034 0.181883423 253 V E 0.283192966 0.158310209 745 A D 0.283094632 0.139036808 739 R S 0.283000418 0.086394522 262 A D 0.282981572 0.21883829 75 E D 0.282861668 0.096240394 122 L V 0.28282995 0.142431105 427 K R 0.282689541 0.126741896 472 K E 0.282354225 0.243592384 69 L V 0.282311609 0.233097353 128 A D 0.282136746 0.144684711 240 L P 0.282112821 0.187484636 840 N D 0.28205862 0.169019904 496 I L 0.281766947 0.156440465 713 R [stop] 0.281751627 0.150509506 759 R I 0.281715415 0.207490665 103 A D 0.281654023 0.156258821 352 K R 0.281644749 0.090972271 23 G D 0.281613067 0.110087313 490 R I 0.28158749 0.189681 534 Y C 0.281578683 0.19797794 728 N K 0.281567938 0.122533743 218 S G 0.28156304 0.0827746 131 Q K 0.28143462 0.261996702 117 D Y 0.281261616 0.150312544 809 C S 0.281246687 0.119977311 899 R S 0.281103794 0.115069396 192 A P 0.281083951 0.125030936 913 N S 0.280977138 0.259159821 232 C S 0.28083211 0.170644437 928 I L 0.280808974 0.249623753 495 A G 0.280579997 0.166279564 917 — ETHAA (SEQ ID NO: 0.280544768 0.259917773 3728) 85 W- LS 0.280472053 0.101385815 344 W [stop] 0.280246002 0.139860723 493 P H 0.280219202 0.225933372 189 G A 0.28010846 0.181165246 565 E G 0.28010846 0.126376781 944 Q R 0.279992746 0.221800854 674 G A 0.27982066 0.112736684 45 E V 0.279758496 0.126165976 281 P A 0.27973122 0.169207983 828 L P 0.279653349 0.165044194 460 A D 0.27950426 0.185233285 539 K R 0.279423784 0.231876099 62 S G 0.279325036 0.105769252 883 S T 0.278909433 0.17133128 166 — LIL 0.27890183 0.114735325 445 D N 0.27879.438 0.120139275 121 R G 0.278752599 0.152495589 66 LN PV 0.278503247 0.058556198 603 — LETGSLK (SEQ ID 0.278503247 0.20379117 NO: 3729) 225 G [stop] 0.278489806 0.182580993 175 — EAN 0.278488851 0.117512649 274 A S 0.278435433 0.213434648 870 D G 0.278347965 0.136371883 683 S T 0.278234202 0.119170388 792 P H 0.277909356 0.196357382 18 N R 0.277904726 0.144376969 484 K R 0.277812806 0.156918996 51 P H 0.27780081 0.207949147 549 A D 0.277618034 0.184792104 285 H Q 0.277595201 0.164383067 772 E [stop] 0.277569205 0.252009775 233 M T 0.277522281 0.101460422 677 — LSRFKDS (SEQ ID 0.277439144 0.176461932 NO: 3730) 444 E D 0.277438575 0.185715982 287 K R 0.277424076 0.122002352 86 E Q 0.277422525 0.267475322 650 K R 0.277338051 0.1661601 119 N K 0.2772012 0.097660237 419 E D 0.27717758 0.091079949 849 Q H 0.277146577 0.10057266 745 A P 0.277094424 0.180486538 895 L V 0.277059576 0.147621158 200 V R 0.276947529 0.109871945 491 G A 0.276923451 0.236639042 437 L P 0.276817656 0.127643327 794 K E 0.276808052 0.108760175 553 N K 0.276534729 0.129122139 500 N K 0.276479484 0.0753.42066 796 Y [stop] 0.276459628 0.151040972 313 K E 0.276424062 0.141250225 184 S R 0.276360484 0.093462218 770 M V 0.276349013 0.177344184 30 T S 0.27626759 0.074607362 887 G C 0.276203171 0.205245818 885 T S 0.276162821 0.125136939 372 K E 0.2761455 0.186164615 161 S F 0.276099268 0.101256778 280 LP PV 0.2760948 0.15312325 118 G A 0.276069076 0.158472607 945 T S 0.275967844 0.217091948 597 W S 0.275959763 0.205648781 700 K [stop] 0.275943939 0.231744011 654 L M 0.275895098 0.222206287 34 R I 0.275728667 0.262529033 650 K N 0.275727906 0.092682765 347 V D 0.275634849 0.162043607 701 Q E 0.275445666 0.129639485 221 S P 0.275424064 0.253543179 902 H Y 0.275413846 0.238626124 408 K N 0.275278915 0.187758493 410 G R 0.275207307 0.148329245 202 R T 0.27519939 0.225294793 190 Q H 0.275101911 0.155497318 296 V A 0.274868513 0.216028266 176 A V 0.274754076 0.101747221 16 D V 0.274707044 0.080710216 338 A G 0.274649181 0.21549192 908 K [stop] 0.274631009 0.235774306 745 A I 0.274596368 0.139876086 582 I I 0.274539152 0.136455089 73 Y H 0.274522926 0.183155681 609 K E 0.274518342 0.096584602 148 — GKPHT (SEQ ID NO: 0.274483854 0.138944547 3731) I 0.274483065 0.167999753 600 L P 0.274446407 0.156944314 609 K N 0.274296988 0.098675974 548 E G 0.274291628 0.174184065 282 P R 0.274223113 0.269615449 743 Y N 0.274041951 0.169744437 273 LA PV 0.273953381 0.083004597 241 — IKYQD (SEQ ID NO: 0.273953381 0.041697608 3732) 752 LI PV 0.273953381 0.179521275 500 — NSILD (SEQ ID NO: 0.273953381 0.096079618 3733) 88 EQ DR 0.273953381 0.132934109 548 E K 0.273785339 0.140999456 758 S T 0.273170088 0.17814745 884 W S 0.27315778 0.127540825 258 E D 0.273147573 0.172394328 720 R M 0.272984313 0.209562405 217 N H 0.272871217 0.212149421 0 M R 0.272866831 0.105028991 376 A G 0.27284261 0.107816996 221 S C 0.272816553 0.204562414 691 LR PV 0.272779276 0.168092844 796 YL DR 0.272779276 0.144849416 439 — EERR (SEQ ID NO: 0.272779276 0.117493254 3734) 383 S N 0.272651878 0.203030872 603 L M 0.272615876 0.2046327 183 Y H 0.27230417 0.167987777 858 R K 0.272264159 0.162833579 525 — KLNLYL (SEQ ID 0.272179534 0.127115618 NO: 3735) 178 D H 0.27217863 0.114858223 186 G S 0.272004663 0.206440397 797 LS PV 0.271846299 0.116235959 434 H L 0.271775834 0.108387354 124 S C 0.271634239 0.201362524 687 — PTH (SEQ ID NO: 0.271046382 0.217907583 3736) 626 R I 0.271037385 0.191496316 717 G V 0.271024109 0.162847575 534 Y [stop] 0.270681224 0.104188898 150 P H 0.270599643 0.192362809 552 A S 0.270597368 0.181876059 150 P S 0.270581156 0.14794261 270 A S 0.270550408 0.145246028 563 S Y 0.270533409 0.17681632 664 — PAV 0.270462826 0.090794222 97 S I 0.270410385 0.155670382 64 A D 0.270367942 0.13574281 143 Q E 0.27021122 0.220203083 686 N I 0.270089028 0.228432562 544 K [stop] 0.270051777 0.124983342 537 G A 0.270050779 0.18424231 902 H L 0.269853978 0.238618549 361 G A 0.269774718 0.191146018 963 S C 0.269617744 0.20243244 965 Y H 0.26944455 0.246260675 66 — LNK 0.269318761 0.181427468 959 — ETWQS (SEQ ID 0.269318761 0.133778085 NO: 3737) 509 — SKQYN (SEQ ID NO: 0.269239232 0.199612231 3738) 32 L I 0.269033673 0.109933858 209 K N 0.269020729 0.109971766 48 R [stop] 0.268939151 0.082435645 466 — T 0.268825688 0.095723888 45 E Q 0.268733142 0.139266278 813 E Q 0.268599201 0.195661988 643 V L 0.268577714 0.156052892 285 H R 0.268299231 0.21489701 317 D G 0.268047511 0.116283826 195 F L 0.268015884 0.108480308 590 R K 0.267781681 0.208536761 180 L V 0.267694655 0.240305187 21 KA TV 0.267470584 0.147038119 210 P H 0.267434518 0.190772597 612 N S 0.267119306 0.129882451 440 E G 0.267419306 0.166870392 651 P L 0.267350724 0.179171164 686 — NPTHILR (SEQ ID 0.267281547 0.145940038 NO: 3739) 56 Q E 0.267209421 0.156465006 656 G D 0.267197717 0.143131022 591 Q E 0.267046259 0.172628923 771 A P 0.266971248 0.20146384 667 I N 0.266893998 0.140849994 333 L P 0.26683779 0.202160591 168 L V 0.266833554 0.09646076 43 R R 0.266528412 0.166392391 76 M T 0.26642278 0.06437874 85 WE CC 0.266335966 0.095081027 784 A D 0.266225364 0.186318048 179 E G 0.266200643 0.159572948 282 P T 0.266142294 0.234821238 505 I V 0.266033676 0.153318009 884 W C 0.265892315 0.146379991 705 Q L 0.265873279 0.218762249 913 N I 0.265873279 0.228181021 775 V S 0.265844485 0.132207982 678 S R 0.265770435 0.147977027 602 S R 0.265750704 0.118408744 121 R T 0.265718915 0.126781949 818 S R 0.265623217 0.145609734 798 S C 0.265584497 0.073889024 864 — DLSVEL (SEQ ID 0.265506357 0.19885122 NO: 3740) 373 R G 0.265364174 0.162678423 803 Q E 0.265269725 0.202509841 628 D E 0.265261641 0.142156395 194 D N 0.265249363 0.155857424 336 R I 0.2651284 0.181377392 602 S I 0.265065039 0.204267576 34 R S 0.265026085 0.223416007 775 y N 0.264899495 0.150356822 647 — SNIK (SEQ ID NO: 0.264896362 0.152108713 3741) 369 A G 0.264866639 0.127314344 407 KKHGEDWG RSTARTGA (SEQ ID 0.26465494 0.11425501 (SEQ ID NO: NO: 3743) 3742) 117 D H 0.264598341 0.092643909 149 K R 0.26429667 0.254633892 624 R S 0.264277774 0.09593797 526 L M 0.26419728 0.176624184 671 D N 0.264084519 0.212711081 572 N K 0.264075863 0.218490453 949 T S 0.263657544 0.110498861 20 KKA T-V 0.263583848 0.126615658 56 Q R 0.263561421 0.151855491 492 K N 0.263524564 0.121563708 315 G D 0.26350398 0.250984577 625 I S 0.263431268 0.11997699 657 I S 0.26332391 0.140695845 688 T R 0.26332192 0.129910161 835 W R 0.263224631 0.136063076 876 S T 0.262876961 0.112192073 468 K R 0.262863102 0.120169191 590 — RQG 0.26279648 0.125412364 912 L R 0.262679132 0.194562045 222 G R 0.262575495 0.121179798 379 P A 0.262556362 0.200217288 7 N Y 0.262545332 0.249153444 514 C R 0.262528328 0.153764358 964 — FY 0.262491519 0.18918584 951 N I 0.262433241 0.181173796 738 A S 0.262344275 0.213159289 109 Q K 0.262161279 0.235829587 371 Y C 0.262089785 0.121531872 62 S I 0.262062515 0.217469036 967 K N 0.261999761 0.11991933 395 R I 0.261975414 0.202071604 546 K E 0.261933935 0.196957538 473 D H 0.26183541 0.210514432 422 — ERIDKIN (SEQ ID 0.261766763 0.175889641 NO: 3744) 661 E D 0.261685468 0.21738252 807 K N 0.261631077 0.137745855 495 A P 0.261336035 0.145111761 474 E V 0.261129255 0.1424745 100 A V 0.261042682 0.097040591 660 G A 0.260992911 0.257791059 613 G V 0.260991628 0.142830183 356 — EKK 0.260606313 0.08939761 419 E R 0.260606313 0.127113021 876 S T 0.262876961 0.112192073 440 E [stop] 0.260572941 0.226197983 245 D Y 0.260411841 0.171518027 838 T A 0.260310871 0.127668195 510 K E 0.260303511 0.170827119 885 T I 0.260229119 0.18213929 606 G C 0.260187776 0.249968408 298 A P 0.260175418 0.137767012 31 L R 0.260094537 0.205569477 19 T I 0.259989986 0.207028692 886 K R 0.259901164 0.087667222 817 T S 0.259831477 0.054519088 901 S T 0.259815097 0.082797155 343 W S 0.259761267 0.144643456 25 T R 0.259617038 0.188030957 238 S P 0.259597922 0.12796144 343 W R 0.259570669 0.092335686 317 D Y 0.259540606 0.174340169 347 — VCNVKK (SEQ ID 0.259425173 0.186479916 NO: 3745) 606 G S 0.259379927 0.201078104 879 N S 0.259300679 0.19356618 784 A S 0.259182688 0.192685039 48 R I 0.259088713 0.132594855 112 L M 0.25908476 0.122948809 181 V A 0.259030426 0.153412207 567 V M 0.258972858 0.206147057 787 A P 0.258909575 0.199316536 741 — LLY 0.258835623 0.170116186 280 — LP 0.258711013 0.142341042 639 — ERREVLD (SEQ ID 0.258711013 0.096645952 NO: 3746) 11 RR AS 0.258711013 0.198257452 660 G V 0.258707306 0.163939116 62 S N 0.258582734 0.206139171 716 G C 0.258579754 0.205579693 185 L M 0.258521471 0.171738368 407 K N 0.258498581 0.130697064 973 W C 0.258383156 0.162271324 419 E [stop] 0.258326013 0.179526252 457 R K 0.25832368.4 0.189885325 876 S R 0.258284608 0.118534232 19 T S 0.258270715 0.163493921 680 F S 0.258237866 0.129529513 2 E A 0.257800465 0.161538463 20 K D 0.257606921 0.080857215 481 K E 0.257527339 0.131433394 227 A P 0.257425537 0.162403215 319 A A 0.25734846 0.183688663 773 R I 0.257312824 0.076585471 59 S R 0.257311236 0.098683009 522 G D 0.257141461 0.205906219 164 E D 0.257089377 0.152824439 705 QA R- 0.257083631 0.186668119 82 H Y 0.256846745 0.145259346 606 G R 0.256772211 0.222683526 281 P L 0.256724807 0.103452649 471 D Y 0.256649107 0.251689277 231 A S 0.256583564 0.187236499 433 K N 0.256518065 0.138408672 883 S G 0.256375244 0.115658726 672 P A 0.256302042 0.169194225 681 KD R- 0.256180855 0.206050883 762 G A 0.256159485 0.149790153 774 Q R 0.256113556 0.176872341 630 P T 0.255980317 0.147464802 151 H Q 0.255948941 0.118092357 38 PDL LT[stop] 0.255810824 0.132108929 240 LT PV 0.255810824 0.138991378 519 — QKDGVK (SEQ ID 0.255711118 0.090066635 NO: 3747) 977 V E 0.255573788 0.223531947 448 S P 0.255534334 0.216106849 872 — LSEE (SEQ ID NO: 0.255312236 0.130213196 3748) 534 -Y DS 0.255312236 0.080703663 765 — GK 0.255312236 0.10865158 28 MK C- 0.255312236 0.091611028 826 EK DR 0.255312236 0.103881802 302 I S 0.2552956 0.169641843 866 S I 0.255156321 0.209048192 472 K M 0.255025429 0.186702335 165 R S 0.25497678 0.100932181 242 K R 0.254948866 0.230748057 311 — KLK 0.25494628 0.09906032 200 V E 0.254874846 0.123567532 129 C R 0.25474894 0.168215252 284 P A 0.254723328 0.141080203 232 — CMG 0.254645266 0.200305653 946 N S 0.2545847 0.199844301 80 I V 0.254434146 0.224490053 327 G V 0.25442364 0.168129037 107 I V 0.254364427 0.144921072 777 R I 0.254281708 0.219559132 801 L P 0.254280774 0.139428109 417 Y H 0.254230823 0.102936144 251 Q L 0.254085129 0.154282551 856 Y [stop] 0.254033585 0.087466157 753 I F 0.25397349 0.160875608 303 W G 0.253842324 0.162875151 852 Y H 0.253666441 0.130229811 223 P S 0.253640033 0.10193396 472 K [stop] 0.253606489 0.18360472 851 T S 0.25343316 0.097399235 725 K E 0.253359857 0.175271591 115 V L 0.253354021 0.093695173 918 T I 0.253156435 0.23080792 630 P L 0.252953716 0.223745102 75 E Q 0.252809731 0.120415311 480 L M 0.252718021 0.192126204 197 S T 0.252713621 0.125864993 779 E Q 0.25259488 0.11277405 340 EV DC 0.252472535 0.047624791 12 R K 0.252469729 0.189301078 515 A S 0.252433747 0.168422609 615 — VIEK (SEQ ID NO: 0.252369421 0.112001396 3749) 513 N S 0.252353713 0.094778563 274 A P 0.252335379 0.222801897 474 E Q 0.252314637 0.161495393 898 K E 0.252289386 0.197783073 397 Q K 0.252164481 0.217428232 455 W S 0.25204917 0.248519347 135 P S 0.252041319 0.143618662 500 N D 0.252036438 0.129905572 204 S I 0.252028425 0.131493678 235 A T 0.251989659 0.158776047 839 I M 0.251899392 0.164461403 473 D N 0.251700557 0.215226558 715 A D 0.251688144 0.14707302 352 K E 0.251658395 0.165058904 423 R I 0.251517421 0.230382833 272 G R 0.251488679 0.185835986 647 S R 0.251423405 0.100129809 333 L M 0.251344003 0.196286065 964 F Y 0.25104576 0.166483614 474 E K 0.250927827 0.172968831 751 M V 0.250846737 0.147715329 471 D N 0.250823008 0.230246417 714 R [stop] 0.250772621 0.098784657 192 A S 0.25063862 0.18266448 668 A D 0.250605134 0.186660163 147 — KG 0.250457437 0.166419391 464 IE DR 0.250457437 0.129773988 325 — LK 0.250457437 0.197198993 812 C R 0.250440238 0.175896886 215 G C 0.250425413 0.161826099 564 G D 0.250350924 0.110254953 787 A D 0.250325364 0.160958271 674 G V 0.25029228 0.086627759 182 T A 0.250160953 0.131790182 383 S R 0.250148943 0.108851149 497 E G 0.250036476 0.073841396 154 Y C 0.250036476 0.229055007 827 K R 0.250016633 0.209047833 722 Y [stop] 0.249927847 0.149439604 380 Y H 0.2.49902562 0.080398395 68 K [stop] 0.249695921 0.134323821 178 D Y 0.24960373 0.233005696 880 D V 0.249521617 0.133706258 543 K R 0.249512007 0.164262829 101 E E 0.249509933 0.220597507 261 L P 0.249467079 0.135680009 410 G A 0.249451996 0.157770206 916 — FETHAAEQA (SEQ 0.249445316 0.231377364 ID NO: 3750) 467 L M 0.249366626 0.154018589 745 A V 0.249363082 0.18169323 773 R K 0.249259705 0.143796066 221 S Y 0.249177365 0.225580403 953 DK CL 0.248980289 0.153230139 213 — QIGGNS (SEQ ID 0.248980289 0.134226006 NO: 3751) 57 P H 0.248900571 0.215896368 301 V L 0.24886944 0.106508651 586 A P 0.248863678 0.211216154 909 F Y 0.248749713 0.182356511 626 R T 0.248743703 0.208846467 186 G R 0.24871786 0.199871451 645 D N 0.248657263 0.126033155 173 K R 0.24855018 0.153000538 519 Q [stop] 0.248535487 0.209163595 888 R I 0.248471987 0.104169936 491 G C 0.248444417 0.204717262 527 N K 0.248397784 0.121054149 893 L V 0.248370955 0.162725859 379 P H 0.248321642 0.237522233 900 F L 0.248316685 0.187112489 974 — KPAV (SEQ ID NO: 0.24830974 0.09950399 3752)[stop] 409 H R 0.248289463 0.198716638 278 I T 0.248133293 0.145997719 230 — DACMG (SEQ ID 0.248087937 0.141736439 NO: 3753) 412 — DWGKVY (SEQ ID 0.248000785 0.085936492 NO: 3754) 135 P H 0.247697198 0.24068468 824 V E 0.247676063 0.211426874 250 H N 0.247644364 0.173527273 101 Q [stop] 0.247598429 0.141658982 364 F S 0.247520151 0.139448351 420 A G 0.247498728 0.234162787 29 KT NC 0.247444507 0.126896702 777 R G 0.247073817 0.140696212 720 R T 0.246870637 0.139065914 529 — YLI 0.246804685 0.066320143 977 V M 0.24675063 0.232768749 414 G C 0.246666689 0.173156358 487 G D 0.246317089 0.205561043 696 S G 0.246296346 0.111834798 515 A G 0.246293045 0.17108612 438 — EE 0.246243471 0.172505379 730 A S 0.246013083 0.141113967 574 N D 0.245981475 0.227302881 747 T S 0.245965899 0.17316365 740 D Y 0.245945789 0.167910919 640 R I 0.245900817 0.188813199 3 I F 0.245678 0.179390362 355 N D 0.245670687 0.09594124 371 Y [stop] 0.245500092 0.105713424 51 P S 0.24544462 0.203086773 28 M L 0.245403036 0.189135882 458 A D 0.245377197 0.208634207 572 N I 0.24524576 0.164550203 959 E [stop] 0.245144817 0.219795779 527 N S 0.245098015 0.16437657 321 P S 0.245086017 0.160736605 579 N K 0.244981546 0.165374413 707 A P 0.244857358 0.22019856 414 G A 0.244717702 0.113316145 548 E V 0.244464905 0.11615159 963 S G 0.244450471 0.188301401 108 D H 0.244382837 0.099322593 19 T R 0.244301214 0.22638105 457 R S 0.244059876 0.203207391 735 R Q 0.243928198 0.170841115 280 L P 0.243719915 0.122012762 627 Q P 0.243601279 0.172067752 571 — VN 0.243561744 0.078796567 25 T A 0.243399906 0.118102255 129 C S 0.243399597 0.045331126 522 G S 0.243323907 0.089702225 695 P K 0.243320032 0.148139423 603 L V 0.243217969 0.148743728 404 H Q 0.242964457 0.173626579 469 E Q 0.242802772 0.126770274 484 KWY NSS 0.242735572 0.182387025 797 L V 0.2425558 0.204091719 928 I F 0.242416049 0.232458614 974 K R 0.242320513 0.114367362 687 P L 0.242304633 0.20007901 885 T R 0.242245862 0.204992576 768 T S 0.242193729 0.178836886 588 — GKRQ (SEQ ID NO: 0.242084293 0.124769338 3755) 262 — ANLKDI (SEQ ID 0.242084293 0.137081914 NO: 3756) 246 I C 0.242084293 0.107590717 288 E [stop] 0.242056668 0.219648186 978 -[stop] YV 0.242009218 0.097706533 110 R [stop] 0.241965346 0.120709959 741 L M 0.241912289 0.193137515 72 D Y 0.241758248 0.224435844 653 N Y 0.24166971 0.0887834 324 R [stop] 0.241651421 0.106997792 293 Y D 0.241440886 0.202068751 695 E A 0.241330438 0.115436697 798 — SKTLAQYT (SEQ ID 0.241309883 0.196326087 NO: 3757) 866 S G 0.241237257 0.109329768 529 V C 0.241113191 0.148105236 102 P S 0.241100901 0.126616893 568 P R 0.241086845 0.174639843 416 V L 0.24098406 0.086334529 834 G S 0.240965197 0.161966438 322 L M 0.240965197 0.161073617 538 G S 0.2.40933783 0.072861862 536 K E 0.240888218 0.130971778 676 P S 0.240757682 0.111329254 108 D E 0.240718917 0.12602791 217 N K 0.240713475 0.15867648 342 D E 0.24062135 0.069616641 471 D H 0.240564636 0.181535186 218 S N 0.240529528 0.151826239 191 R I 0.240513696 0.229207246 963 — SFY 0.240421887 0.098315268 77 K N 0.240381155 0.116252284 637 — TFER (SEQ ID NO: 0.240288787 0.148900082 3758) 571 V L 0.240279118 0.074639743 346 M T 0.240147015 0.108146398 512 Y [stop] 0.240104852 0.068415116 430 G C 0.240047705 0.20806366 599 D G 0.239869359 0.206138755 462 F S 0.23971457 0.144092402 724 S R 0.239681347 0.127922837 61 T S 0.239626948 0.164373644 525 K [stop] 0.239380142 0.131802154 296 V E 0.239355864 0.120748179 968 K Q 0.238999998 0.129755167 617 E K 0.238964823 0.084548152 120 E K 0.238945442 0.100801456 44 L V 0.238860984 0.10949901 315 G R 0.238751925 0.215543005 87 E [stop] 0.238731064 0.177299521 818 S G 0.238509249 0.201919192 189 G V 0.238447609 0.179422249 394 A D 0.238439863 0.125867824 861 — V 0.238439176 0.202222792 357 K E 0.238434177 0.184905545 353 L V 0.23831895 0.17206072 488 D V 0.2382354 0.188903119 684 — LGNPT (SEQ ID NO: 0.2382268 0.157487774 3759) 376 A V 0.238191318 0.142572457 349 N D 0.238174065 0.053089179 331 F S 0.238131141 0.093269792 971 E D 0.238076025 0.194709418 775 Y F 0.238057448 0.214475137 730 A I 0.238038323 0.175731569 631 — ALF 0.237949975 0.190053084 504 D H 0.23794567 0.139048842 94 G D 0.237937578 0.15570335 291 E [stop] 0.237828954 0.19900832 871 R I 0.237759309 0.236033629 761 F V 0.237669703 0.128380283 910 — VCLN (SEQ ID NO: 0.237633429 0.152561858 3760) 731 D Y 0.237566392 0.167223625 245 D A 0.237553897 0.189220496 979 L-E VWS 0.237546222 0.150693183 208 V E 0.237546113 0.17752812 483 Q R 0.23746372 0.159123209 634 V M 0.237398857 0.152995502 837 T I 0.237183554 0.104666535 479 E Q 0.237085358 0.157162064 555 F V 0.237065318 0.182110462 872 LS PV 0.23698628 0.179042308 601 L P 0.236954247 0.122470012 127 F L 0.236892252 0.129435749 204 S C 0.236855446 0.164372504 82 H Q 0.236837713 0.172606609 861 — VVKDLSVE (SEQ ID 0.236770505 0.195127344 NO: 3761) 493 P L 0.236700832 0.181806123 474 E G 0.236695789 0.180206764 302 I F 0.236588615 0.136160472 109 Q R 0.236576305 0.166840659 97 S R 0.236508024 0.179878709 40 L V 0.236210141 0.21459356 761 F C 0.236145536 0.170046245 50 K N 0.236137845 0.22219675 205 N K 0.236073257 0.12180008 399 G D 0.236045787 0.181873656 521 S Y 0.235934057 0.180076567 665 A D 0.235822456 0.220273467 252 K R 0.235675801 0.120466673 646 S R 0.235675637 0.183914638 102 P A 0.235653058 0.16760539 810 S N 0.235539825 0.164257896 936 R S 0.235496123 0.188093786 111 K R 0.235492778 0.118354865 220 A V 0.235467868 0.198253635 855 — RYK 0.235222552 0.156668306 354 I N 0.235178848 0.098023234 158 C F 0.235135625 0.169427052 689 H R 0.235102048 0.220671524 594 E-F GRII (SEQ ID NO: 0.235051862 0.132444365 3762) 154 Y D 0.234980588 0.232501764 870 D V 0.234951394 0.118777361 198 I N 0.234906329 0.184047389 76 M I 0.234796263 0.126238567 434 H N 0.234726089 0.143174214 484 -KW NSSL (SEQ ID NO: 0.234680329 0.165662856 3763) 49 K [stop] 0.234415257 0.114263318 896 L P 0.234287413 0.192149813 530 L V 0.234192802 0.173965176 643 V A 0.234106948 0.176627185 711 E K 0.234002178 0.154011045 918 — THAAEQ (SEQ ID 0.23373891 0.117744474 NO: 3764) 473 D E 0.233630727 0.181285916 666 V E 0.233615017 0.210063502 610 — LANGRVIE (SEQ ID 0.233598549 0.098900798 NO: 3765) 463 V A 0.233582437 0.13705941 771 A V 0.233335501 0.144017771 89 Q H 0.233314663 0.120225936 18 N D 0.233234266 0.100130745 547 P A 0.233232691 0.192665943 628 D H 0.233191566 0.113338873 290 I V 0.233178351 0.147527858 837 — TTIN (SEQ ID NO: 0.233038063 0.141130326 3766) 909 — FV 0.233038063 0.131142006 260 R G 0.232970656 0.120191772 707 — AKEVEQR (SEQ ID 0.232896265 0.116012039 NO: 3767) 638 F S 0.232893598 0.149395863 671 D A 0.232880356 0.163658679 443 S T 0.232784832 0.170920909 392 K N 0.232687633 0.108105318 500 N I 0.232640715 0.1305158 111 K E 0.232613623 0.097737029 570 E Q 0.232497705 0.099759258 645 D E 0.2323596 0.127143455 54 I N 0.23228755 0.182788712 725 K R 0.232253631 0.11253677 771 A S 0.232158252 0.16845905 896 L V 0.232108864 0.141878039 487 G V 0.232053935 0.22651513 655 I V 0.231994505 0.148078533 708 K R 0.231988811 0.183732743 699 E D 0.231934703 0.178386576 446 A P 0.231896096 0.131534649 902 H P 0.231793863 0.226418313 555 F S 0.231772683 0.154329003 685 G R 0.231646911 0.113490558 430 G A 0.231581897 0.168869877 423 R G 0.231294589 0.188648387 773 R S 0.231238362 0.139470334 148 — GKP 0.231166477 0.084708483 795 TY PG 0.231166477 0.229360354 598 N S 0.230890539 0.114382772 109 Q [stop] 0.230738213 0.089332392 481 — KLQK (SEQ ID NO: 0.23071553 0.20441951 3768) 592 -GR DNQ 0.230655892 0.071944702 254 I T 0.2306357 0.069580284 530 L R 0.230571343 0.193066361 365 W [stop] 0.230333383 0.12753339 131 Q R 0.2302555 0.206903114 244 Q E 0.230190451 0.222512927 900 F I 0.230181139 0.149890666 318 E Q 0.230160478 0.212890421 312 L M 0.230110955 0.204915228 106 N S 0.230101564 0.155287559 968 K R 0.230017803 0.168949701 631 A P 0.229723383 0.159718894 610 L V 0.229644521 0.180175813 847 E G 0.229640073 0.111868196 636 — LT 0.229485665 0.192188426 665 A G 0.229408129 0.212381399 82 H R 0.229295108 0.108155794 371 Y D 0.229277426 0.117283148 148 G V 0.229238098 0.159823444 443 S I 0.229142738 0.169822985 660 G C 0.229029418 0.194710612 181 V D 0.228966959 0.164951106 832 A P 0.228767879 0.092204547 152 I A 0.228705386 0.182569685 685 G A 0.228675631 0.17392363 112 L P 0.22866263 0.221195984 214 I T 0.22857342 0.11423526 610 L M 0.22841473 0.205382368 110 R G 0.228257249 0.086720324 590 R S 0.228041456 0.143022556 596 I M 0.227907909 0.117874099 1 Q P 0.227785203 0.168369144 567 V E 0.227660557 0.156302233 32 L V 0.227635279 0.12966479 65 N S 0.22749218 0.063907676 291 E G 0.227296993 0.128103388 635 A V 0.22713711 0.159876533 894 S I 0.227093532 0.165363718 675 C R 0.227077437 0.19145584 863 K E 0.227027728 0.176903569 130 S N 0.226933191 0.162445952 187 K E 0.226883263 0.185467572 330 S G 0.226753105 0.138020012 224 V A 0.226536103 0.153342124 802 A I 0.226368502 0.154358709 148 G S 0.226168476 0.097680006 732 D E 0.226134547 0.109002487 864 D G 0.226094276 0.177950676 140 K R 0.226067524 0.114127554 814 F S 0.225959256 0.114511043 215 G D 0.225350951 0.086324983 138 V L 0.225143743 0.155359682 192 A T 0.22512485 0.144695235 502 I S 0.225038868 0.197567126 494 F V 0.224968248 0.143764694 162 E D 0.224950043 0.153078143 788 Y [stop] 0.22492674 0.129943744 263 N I 0.22.4722541 0.117014395 918 — THAAEQA (SEQ ID 0.224719714 0.202778103 NO: 3769) 272 G A 0.224696933 0.211543463 322 L V 0.22.46772 0.156881144 132 C R 0.224659007 0.146010501 657 I F 0.224649177 0.161870244 917 — E 0.224592553 0.150266826 704 — IQAAKE (SEQ ID 0.224567514 0.109443666 NO: 3770) 328 — FPS 0.224567514 0.088644166 455 W R 0.224240948 0.159412878 528 — LY 0.224210461 0.204469226 289 G A 0.224158556 0.07475664 477 RCE SFS 0.224109734 0.175971589 290 I M 0.224106784 0.121750806 699 EK AV 0.223971566 0.120407858 190 — QRALDFY (SEQ ID 0.223971566 0.118248938 NO: 3771) 287 K [stop] 0.223966216 0.119362605 33 V A 0.223884337 0.200194354 321 P R 0.223833871 0.153353055 350 V L 0.223803585 0.123552417 598 N D 0.223755594 0.127015451 784 A V 0.22374846 0.140061096 540 L P 0.223660834 0.130300184 330 S R 0.2236138 0.142019721 162 E Q 0.223613045 0.201165398 128 A V 0.223401934 0.126557909 296 V L 0.223401818 0.13392173 634 V E 0.223309652 0.118175475 356 E Q 0.22323735 0.143945409 289 G V 0.223202197 0.145913012 805 T N 0.223188037 0.139245678 599 D Y 0.223008187 0.183323322 246 I M 0.222998811 0.092368092 36 M K 0.222893666 0.113406903 476 C [stop] 0.222743024 0.176188321 464 I V 0.222701858 0.18421718 224 V L 0.222626458 0.136476862 42 E G 0.22255062 0.189996134 832 A S 0.222538216 0.190249328 734 V I 0.222476682 0.141366416 146 D H 0.22246095 0.16577062 755 AN DS 0.222404547 0.10970681 581 I V 0.222357666 0.17105795 698 K [stop] 0.222296953 0.103211977 507 G D 0.22225927 0.153400026 246 I V 0.222098073 0.120973819 47 L P 0.222066189 0.162841956 301 VI CL 0.222059585 0.122617461 210 PL DR 0.222059585 0.108090576 174 — PEANDE (SEQ ID 0.222059585 0.182232379 NO: 3772) 160 — VSE 0.222059585 0.137662445 68 K E 0.222044865 0.16348242 119 K [stop] 0.221989288 0.160692576 230 — DAC 0.221929991 0.119956442 559 -I TV 0.221929991 0.162385076 125 S T 0.221924231 0.192354491 738 A P 0.221764129 0.166374434 389 K L 0.221512528 0.096823472 829 K M 0.22130603 0.111760034 435 I V 0.221227154 0.143247597 626 R S 0.221038435 0.198631408 135 P R 0.221017429 0.116069626 203 E Q 0.22076143 0.119826394 783 T I 0.220740744 0.134860122 672 P S 0.220729114 0.141569742 361 G D 0.220639166 0.141910298 690 I M 0.220631897 0.180897111 552 A G 0.220614882 0.110523427 441 R I 0.220543521 0.155159451 218 S R 0.220420945 0.153071166 917 — ETHAAE (SEQ ID 0.220288736 0.09840913 NO: 3773) 204 S R 0.220214876 0.101819626 255 K E 0.220080844 0.12573371 479 E D 0.220079089 0.099777598 438 E G 0.219979549 0.120742867 605 T I 0.219976898 0.126979027 109 Q E 0.219959218 0.140761458 744 Y C 0.219956045 0.132833086 930 — RSWLFL (SEQ ID 0.219822658 0.120132898 NO: 3774) 172 H Q 0.219757029 0.10461302 329 P A 0.219753668 0.110968401 783 T S 0.219504994 0.118049041 610 L P 0.219499239 0.160199117 38 P A 0.219404694 0.107368636 446 A V 0.218887024 0.176662627 41 R K 0.218858764 0.128896181 810 S R 0.21870856 0.129689435 83 V L 0.218625171 0.138945755 474 E D 0.218570822 0.130.400355 712 Q [stop] 0.218254094 0.091444311 371 V H 0.218137961 0.189187449 35 V L 0.218110612 0.095949997 687 P R 0.21806458 0.159278352 621 Y N 0.218036238 0.089590425 753 I N 0.21792347 0.101271232 337 Q L 0.217694196 0.180223104 366 Q E 0.217564323 0.195945495 156 G R 0.217510036 0.186872459 813 G A 0.217404463 0.109971024 911 C W 0.217360044 0.181625646 896 L Q 0.217312492 0.09770592 395 R S 0.217267056 0.103436045 506 S R 0.217238346 0.104753923 459 KA NR 0.217171538 0.126085081 605 T S 0.217140582 0.104288213 147 K R 0.217113942 0.165662771 358 K R 0.217018444 0.148484962 710 V E 0.216906218 0.158321115 948 T N 0.216794988 0.204294035 62 S T 0.216604466 0.167204921 827 K E 0.216603742 0.107241416 457 R G 0.216513116 0.052626339 159 N K 0.216507269 0.109954163 177 N D 0.216431319 0.179290406 921 — AEQAALN (SEQ ID 0.216389396 0.149922966 NO: 3776) 633 — FV 0.216309574 0.179645361 433 — KHI 0.216309574 0.092546366 375 E [stop] 0.216261145 0.199757211 297 V A 0.216143366 0.15509483 148 — GKPHTNYF (SEQ ID 0.216132461 0.211503255 NO: 3777) 645 D V 0.21604012 0.117781298 147 KG R- 0.215998635 0.103939398 292 A S 0.215943856 0.157240024 387 R G 0.215798372 0.151215331 157 R T 0.215790548 0.152247144 203 E K 0.215703649 0.168783031 123 T S 0.21570133 0.105624839 383 S G 0.215603433 0.137401501 310 Q [stop] 0.21551735 0.135329921 592 G A 0.215456343 0.13373272 562 K R 0.215325036 0.122831356 951 N S 0.21531813 0.214926405 823 R I 0.215273573 0.191310901 723 A P 0.215193332 0.108699964 713 R T 0.215008884 0.104394548 878 N I 0.214931515 0.11752804 145 N H 0.214892161 0.185408691 338 A T 0.21480521 0.15310635 169 L V 0.214751891 0.163877193 30 T P 0.214714414 0.144104489 164 E A 0.214693055 0.151150991 734 V F 0.214507965 0.184315198 841 G V 0.21449654 0.163419397 848 G D 0.214491489 0.166744246 93 VGL WA[stop] 0.21434042 0.171341302 747 T K 0.214238165 0.122971462 688 T K 0.214222211 0.126368648 878 N Y 0.214205323 0.111547616 190 Q E 0.214170881 0.122424442 523 — VKKLN (SEQ ID NO: 0.214126014 0.14801882 3778) 792 — PSK 0.214126014 0.088425611 171 — PHK 0.214126014 0.186440571 918 — TH 0.214126014 0.10224323 833 T S 0.214086868 0.0993742 72 D E 0.214062412 0.115630034 560 N K 0.213945541 0.173784949 906 Q L 0.213845132 0.187470303 461 S I 0.21384342 0.180386801 622 N I 0.213809938 0.161761781 768 T I 0.213809607 0.08102538 204 — SNH 0.21345676 0.114570097 944 — Q 0.213449244 0.157411492 49 K R 0.213334728 0.181645679 411 E [stop] 0.213222053 0.149931485 719 S A 0.213134782 0.140566151 731 D E 0.213022905 0.120709041 475 F S 0.213010505 0.137035236 305 N K 0.213008678 0.108878566 30 TL PC 0.212945774 0.075648365 611 A G 0.212935031 0.195766935 266 DI AV 0.212926287 0.127744646 730 — ADDM (SEQ ID NO: 0.212926287 0.097551919 3779) 684 — LG 0.212926287 0.093015719 979 LE[stop]GSPG VSSKDLK (SEQ ID 0.212926287 0.091900005 (SEQ ID NO: NO: 3780) 3668) 241 — TKYQ (SEQ ID NO: 0.212926287 0.1464038 3781) 949 I I 0.212862846 0.194719268 709 E G 0.212846074 0.116849712 926 — LN 0.212734596 0.151263965 901 — SHRPVQE (SEQ ID 0.212684828 0.084903934 NO: 3782) 459 K E 0.212680715 0.093525423 228 L V 0.212591965 0.092947468 831 T I 0.212576099 0.16705965 819 A T 0.212522918 0.164976137 645 D G 0.21251225 0.121902674 794 K R 0.212502396 0.178916123 859 Q P 0.212311083 0.170329714 738 A G 0.212248976 0.161293316 409 H Q 0.212187222 0.201696134 192 — ALDFY (SEQ ID NO: 0.212165997 0.132724298 3783) 782 — LTAKLA (SEQ ID 0.212165997 0.121732843 NO: 3784) 86 EEF DCL 0.212165997 0.090389548 251 Q H 0.212109948 0.151365816 197 S R 0.211641987 0.087103971 196 Y C 0.211596178 0.195825393 125 S I 0.211507893 0.117116373 237 A T 0.211485023 0.118730598 574 N S 0.211257767 0.135650502 73 Y C 0.211200986 0.169366394 380 Y [stop] 0.21093329 0.132735624 219 C Y 0.210905605 0.190298454 777 R S 0.210879382 0.15535129 799 — KTLAQYT (SEQ ID 0.210719207 0.130227708 NO: 3785) 79 A T 0.210637972 0.047863719 654 L R 0.210450467 0.143325776 179 E K 0.210277517 0.147945245 587 F E 0.210211385 0.204490333 414 E Q 0.210197326 0.171958109 546 K Q 0.210196739 0.176398222 645 D Y 0.210085231 0.190055155 67 N S 0.210019556 0.1.3100266 403 L P 0.209919624 0.075615563 452 L P 0.209882094 0.127675947 733 M V 0.209851123 0.136163056 872 L P 0.209831548 0.152338232 882 S R 0.209789855 0.108285285 679 R T 0.209762925 0.169692137 553 — NRFYTVI (SEQ ID 0.209733011 0.13607198 NO: 3786) 650 — KPMN (SEQ ID NO: 0.209706804 0.099600175 3787) 802 AQ DR 0.209706804 0.100831295 415 K R 0.209696722 0.172211853 470 A P 0.209480997 0.11945606 389 K R 0.209459216 0.190864781 233 M K 0.209263613 0.148910419 846 V A 0.209194154 0.132301095 803 Q R 0.209112961 0.157007924 594 -EF GRI 0.209067243 0.142920346 418 D Y 0.208952621 0.201914561 424 I N 0.208940616 0.184257414 152 — TNYFG (SEQ ID NO: 0.208921679 0.069015043 3788) 184 — SLGKFGQ (SEQ ID 0.208921679 0.145515626 NO: 3789) 944 — QTNK (SEQ ID NO: 0.208921679 0.115799997 3790) 435 IK DR 0.208921679 0.100379476 926 LN PV 0.208921679 0.122257143 31 L P 0.208720548 0.120146815 595 F I 0.208631842 0.129889087 765 G R 0.208575469 0.10091353 506 S G 0.208540925 0.155512988 408 K R 0.208534867 0.133392724 171 P A 0.208511912 0.145333852 953 — DK 0.208375969 0.185478366 518 W C 0.208374964 0.121746678 34 R G 0.208371871 0.100655798 663 — IPAV (SEQ ID NO: 0.208314284 0.125213293 3791) 737 T S 0.208225559 0.129504354 6 I N 0.208110644 0.078448603 677 L M 0.208075234 0.142372791 456 L Q 0.208040599 0.142959764 190 Q R 0.207948331 0.189816674 382 S G 0.207889255 0.137324724 953 D H 0.207762178 0.180457041 522 G R 0.207711735 0.201735272 655 I F 0.207554053 0.114186846 345 D N 0.207459671 0.194429167 619 T A 0.20742287 0.107807162 273 L M 0.207369167 0.150911133 695 E G 0.207324806 0.170023455 662 N S 0.207198335 0.146245893 102 P R 0.207103872 0.104479817 212 E G 0.207077093 0.167731322 118 G V 0.20699607 0.113451465 841 G P 0.20698149 0.160303912 501 S R 0.206963691 0.188972116 402 L M 0.206953352 0.103953797 642 — EVLDSSN (SEQ ID 0.206944663 0.088763805 NO: 3792) 426 — KKVEGLS (SEQ ID 0.206944663 0.120828794 NO: 3793) 273 — LA 0.206944663 0.200099204 631 AL DR 0.206944663 0.132545056 75 E V 0.206746722 0.108008381 159 — NVSEHER (SEQ ID 0.206678079 0.108971025 NO: 3794) 974 — K 0.206678079 0.087902725 13 L I 0.206678079 0.17404612 135 P L 0.206613655 0.11493052 576 D N 0.206571359 0.197674836 396 — YQ 0.206474109 0.165665557 426 K R 0.206261752 0.175070461 720 R S 0.206187746 0.130762963 731 D H 0.206140141 0.18515671 792 — PSKTY (SEQ ID NO: 0.206037621 0.119445689 3795) 470 — ADKDEFC (SEQ ID 0.206037621 0.160849031 NO: 3796) 846 — VEGQ (SEQ ID NO: 0.205946011 0.115023996 3797) 730 — ADDMV (SEQ ID 0.205946011 0.203904239 NO: 3798) 195 F S 0.205931771 0.0997168 763 R G 0.205931024 0.177755816 668 A G 0.205831825 0.181720031 123 T I 0.205810457 0.169798366 394 A G 0.205790009 0.129212763 776 T N 0.205770287 0.088016724 779 P D 0.205703015 0.117547264 787 A G 0.205542455 0.113825299 448 S C 0.205480956 0.165327281 341 V L 0.205333121 0.121382241 351 K [stop] 0.205260708 0.137391414 408 K [stop] 0.205233141 0.101895161 626 R [stop] 0.204917321 0.133170214 426 K N 0.204813329 0.115277631 217 N D 0.204605492 0.15571936 55 P A 0.204194052 0.203451056 979 L-E VSSK (SEQ ID NO: 0.204463305 0.104199954 3669) 789 EG GD 0.204429605 0.094907378 174 P H 0.204410022 0.192547659 37 T I 0.20435056 0.108024009 230 D Y 0.204310577 0.163888419 369 A D 0.204246596 0.143255593 567 V L 0.204221782 0.133245956 356 E G 0.204079788 0.096784994 826 E G 0.204045427 0.079692638 234 — GAVASF (SEQ ID 0.203921342 0.148635343 NO: 3936) 791 — LP 0.203921342 0.086381396 550 F Y 0.203856294 0.154808557 139 Y H 0.203748432 0.112669732 842 K E 0.203739019 0.14619773 565 E D 0.203689065 0.115937226 667 IA TV 0.203650432 0.146532587 554 — RFYTV (SEQ ID NO: 0.203650432 0.085651298 4123) 481 — KLQKW (SEQ ID 0.203650432 0.173739202 NO: 4006) 64 A V 0.203579261 0.147026682 429 E K 0.203478388 0.197959656 659 R W 0.203469266 0.155374384 775 Y [stop] 0.203457477 0.112309611 420 A P 0.203276202 0.137871454 844 — LK 0.20327417 0.108693201 543 KK DR 0.20327417 0.081409516 483 QK DR 0.203103924 0.108226373 661 E-N DHSRD (SEQ ID NO: 0.203103924 0.080468187 3886) 591 — QGREFIWN (SEQ ID 0.203103924 0.127711804 NO: 4103) 434 — HIKLE (SEQ ID NO: 0.203103924 0.128782985 3963) 192 A D 0.203101012 0.088663269 979 LE VW 0.203097285 0.114357374 905 V E 0.2029568 0.158582123 648 N K 0.202865781 0.076554962 811 N D 0.202736819 0.184175153 573 F Y 0.202703202 0.143842683 388 K E 0.202623765 0.1173393 265 K [stop] 0.202622408 0.159704419 511 Q E 0.202512176 0.199826141 375 E Q 0.202480508 0.162732896 106 N K 0.202431652 0.125127347 52 E G 0.202421366 0.17180627 597 W [stop] 0.202346989 0.135138719 153 N K 0.202320957 0.084739162 471 D E 0.202309983 0.069685161 486 Y H 0.202105792 0.189019359 732 D V 0.202045584 0.172766987 833 T I 0.202003023 0.114654955 220 A D 0.201986226 0.167650811 386 D G 0.201893421 0.144223833 271 N K 0.201821721 0.136225013 236 VA -C 0.201781577 0.118494484 661 E Q 0.201717523 0.126595353 644 L M 0.201626647 0.191409491 326 K E 0.201516415 0.172628702 584 P T 0.201277532 0.157595812 216 G A 0.201151425 0.135718161 158 C R 0.200895575 0.132515505 557 T P 0.20079665 0.175823626 615 — VIEKTLY (SEQ ID 0.20079665 0.14533527 NO: 4209) 121 R I 0.200425228 0.146944719 67 N K 0.200404848 0.19495599 258 E G 0.200396788 0.144009482 232 — CM 0.200312143 0.13867079 526 — LN 0.200312143 0.15960761 202 -RE SSS 0.200312143 0.113603268 68 K T 0.200238961 0.196349346 448 S Y 0.200204468 0.144800694 837 — TTI 0.200162181 0.089943784 158 — CNVSE (SEQ ID NO: 0.200162181 0.088327822 3872) 796 — YLSKTLA (SEQ ID 0.200048174 0.1285851 NO: 4265) 276 — PK 0.200048174 0.079289415 801 — LAQY (SEQ ID NO: 0.200048174 0.196038539 4027) 651 — PMNLI (SEQ ID NO: 0.200048174 0.135317157 4092) 756 — N 0.200048174 0.172777109 149 KPHTNY (SEQ ID 0.200048174 0.109852809 NO: 4012) 494 — FA 0.200048174 0.123840308 181 V I 0.19996686 0.166465973 616 I M 0.19990025 0.183539616 227 A — 0.199865011 0.119483676 866 S R 0.199834101 0.105100812 664 — PAVIALT (SEQ ID 0.199723054 0.116432821 NO: 4085) 955 R W 0.199719648 0.122422647 507 G A 0.199700659 0.133738835 925 — ALNI (SEQ ID NO: 0.199681554 0.112069534 3855) 419 — EAW 0.199681554 0.151874009 663 I N 0.199667187 0.147345549 845 K R 0.199649448 0.119477749 782 L V 0.199620025 0.156520261 173 K E 0.199587002 0.098249426 615 — VIEKTLYN (SEQ ID 0.199584873 0.182641156 NO: 4210) 630 P A 0.199530215 0.103804567 446 AQ DR 0.199529716 0.10633379 374 Q [stop] 0.199329379 0.131990193 778 M K 0.199291554 0.158456568 858 R S 0.199265103 0.108121324 579 N I 0.19915895 0.103520322 63 R G 0.199095742 0.127135026 646 S I 0.199062518 0.104634011 90 K E 0.199052878 0.198240775 439 E Q 0.198907882 0.179263601 621 Y C 0.198885865 0.125823263 310 Q H 0.198723557 0.146313995 60 N K 0.198659421 0.192782927 299 Q R 0.1986231 0.112149973 203 — ES 0.19897765 0.14607778 279 T S 0.198506775 0.126696973 278 I N 0.198457202 0.188794837 462 — FV 0.198353725 0.132924725 264 — LK 0.198353725 0.107390522 296 — VVAQ (SEQ ID NO: 0.198353725 0.116995821 4249) 152 T I 0.198333224 0.117839718 720 R G 0.198275202 0.180739318 236 V L 0.198162379 0.091047961 903 R [stop] 0.197764314 0.184873287 190 Q [stop] 0.197676182 0.135507554 19 TK PG 0.197606812 0.087295898 554 R [stop] 0.197270424 0.119115645 63 R K 0.197266572 0.156106069 671 D Y 0.197186873 0.193857965 380 YL T [stop] 0.197159823 0.186882164 210 P R 0.197120998 0.088119535 637 T S 0.196993711 0.074085124 657 I M 0.196919314 0.094328263 458 — AK 0.196819897 0.136384351 304 V F 0.196773726 0.171052025 263 N K 0.196728929 0.082781462 601 L V 0.196677335 0.163553469 545 I N 0.196522854 0.15815205 571 VN AV 0.196419899 0.093569564 284 — PHTKE (SEQ ID NO: 0.196419899 0.146831822 4090) 163 -HE PTR 0.196323235 0.180126799 57 P L 0.196165872 0.129483671 659 R P 0.196165872 0.140190097 784 A P 0.196137855 0.183129066 323 Q H 0.196115938 0.150227482 763 R W 0.195967691 0.113028792 257 N Y 0.195936425 0.189617104 125 S G 0.19588405 0.126337645 787 A T 0.195855224 0.170500255 213 Q L 0.195810372 0.164285983 979 — VSS 0.195756097 0.115771783 466 G D 0.195631404 0.128114426 388 K R 0.195529616 0.155892093 767 R K 0.195477683 0.182282632 673 E V 0.195473785 0.111723182 864 D Y 0.195306139 0.092331083 885 I K 0.195258477 0.131521124 856 Y C 0.195214677 0.129834532 205 N S 0.194826059 0.070507132 696 S R 0.194740876 0.106074027 498 A Y 0.194435389 0.108630638 281 P H 0.194325757 0.164586878 106 N D 0.194156411 0.113601316 756 — NLS 0.194120313 0.113317678 591 — QGRE (SEQ ID NO: 0.194120313 0.089464524 4102) 572 N D 0.194049735 0.182872987 762 G S 0.193891502 0.138436771 41 R [stop] 0.193882715 0.149226534 370 G D 0.193873435 0.131402011 58 I T 0.193827338 0.18015548 64 A S 0.193814684 0.163559402 203 E G 0.193809853 0.182009134 318 E K 0.193618764 0.182298755 867 V L 0.193526313 0.149480344 343 W [stop] 0.193259223 0.086409476 920 — AAEQ (SEQ ID NO: 0.1932196 0.09807778 3841) 559 I N 0.193172208 0.185545361 577 D E 0.193102893 0.104761592 721 K N 0.193081281 0.123219324 767 R S 0.19293341 0.180949858 353 L P 0.192916533 0.142447603 662 N D 0.192798707 0.113762689 87 E G 0.192780117 0.1542337 347 V G 0.192656101 0.11936042 440 E Q 0.192625703 0.16228978 698 K N 0.192440231 0.067040488 757 L Q 0.192392703 0.11735809 446 — AQSK (SEQ ID NO: 0.192307738 0.188279486 3862) 91 D Y 0.192222499 0.161107527 65 N K 0.192152721 0.086051749 228 L Q 0.192019982 0.075226208 107 I N 0.191587572 0.153969194 307 N S 0.191540821 0.186358955 944 QT PV 0.191151442 0.133263263 526 — LNLYLI (SEQ ID NO: 0.191451442 0.098341333 4049) 750 -A LS 0.191451442 0.07841082 651 — PMN 0.191451442 0.159749911 370 — GYKRQ (SEQ ID NO: 0.191451442 0.172523736 3959) 654 L V 0.191441378 0.100236525 332 P L 0.191427852 0.132400599 724 S G 0.191322798 0.15242.4888 206 H D 0.191266107 0.183831734 594 E D 0.191101272 0.114552929 525 K E 0.190973602 0.101119046 576 D E 0.190942249 0.134849057 663 I V 0.190923863 0.098130963 225 G A 0.190920356 0.167486936 227 A V 0.190541259 0.158522801 539 — KLRF (SEQ ID NO: 0.190525892 0.118424918 4007) 336 — RQANEVD (SEQ ID 0.190525892 0.095546149 NO: 4133) 511 — QYN 0.190525892 0.10542285 182 — TY 0.190525892 0.095282059 955 R K 0.190477708 0.163763612 669 L V 0.190343627 0.076107876 492 K Q 0.190290589 0.150334427 721 K E 0.190242607 0.123347897 389 K E 0.190239723 0.177951808 619 T I 0.190153498 0.116807589 93 V E 0.190153374 0.163133537 336 R G 0.190122687 0.099072113 481 — KLQ 0.190063819 0.144467422 878 N K 0.190097445 0.16631012 847 — EG 0.190063819 0.165413398 655 I N 0.190024208 0.138898845 696 S- TG 0.189908515 0.068382259 55 P R 0.189907461 0.115309052 269 S N 0.18989023 0.150359662 210 P L 0.189875815 0.142379934 798 S V 0.18982788 0.189131471 258 E K 0.189676636 0.183203558 190 Q P 0.189645523 0.168321089 377 L V 0.189542806 0.136436344 500 N S 0.189535073 0.180860478 295 N S 0.18951855 0.108197323 974 K [stop] 0.189482309 0.139647592 54 I V 0.189429698 0.1555694 736 N D 0.189336313 0.075796871 505 I N 0.189099927 0.151637022 396 Y H 0.189044775 0.129353397 117 D V 0.188915066 0.132090825 8 K M 0.188755388 0.159809948 699 E K 0.188739566 0.092771182 132 C G 0.188700628 0.133537793 338 A V 0.188698117 0.151434141 641 R [stop] 0.188367145 0.11062471 208 V L 0.188333358 0.080207667 207 P T 0.188302368 0.15553127 936 — RSQEYK (SEQ ID 0.188141846 0.120467426 NO: 4140) 428 VE AV 0.188141846 0.111936388 419 — EAWE (SEQ ID NO: 0.188141846 0.161004571 3905) 148 — GKPHTN (SEQ ID 0.188141846 0.126152225 NO: 3947) 972 — VWKPA (SEQ ID 0.188111846 0.100559027 NO: 4251) 328 F S 0.188082476 0.152191585 596 I N 0.188043065 0.141822306 482 L V 0.187880246 0.186391629 582 I V 0.18725447 0.136748728 699 E Q 0.187137878 0.176072109 758 S I 0.18709104 0.158068821 113 I N 0.187005943 0.142849404 968 K E 0.186636923 0.128956962 168 — LLSPH (SEQ ID NO: 0.186576707 0.08269231 4045) 833 TGWM (SEQ ID PAG[stop] 0.186576707 0.125195246 NO: 3832) 272 — GLAFPIK (SEQ ID 0.186576707 0.060722091 NO: 3949) 529 — YLIIN (SEQ ID NO: 0.186576707 0.104569212 4264) 261 — LANLKD (SEQ ID 0.186576707 0.081389931 NO: 4026) 884 W [stop] 0.18656617 0.16960295 719 S F 0.186508523 0.176978743 879 N K 0.186386792 0.12079248 712 Q L 0.186379419 0.129128012 583 L P 0.186146799 0.156442099 323 — QRLK (SEQ ID NO: 0.186069265 0.110701992 4111) 358 — KEDG (SEQ ID NO: 0.18604711 0.119601341 3989) 835 — WM 0.18604741 0.100790291 839 — INGKELK (SEQ ID 0.18604741 0.115878922 NO: 3977) 463 V E 0.186017541 0.06776571 299 Q H 0.185842115 0.085070655 832 A C 0.185822701 0.103905008 127 F Y 0.185786991 0.140080792 159 N S 0.185693031 0.145375399 532 — IN 0.185685948 0.088889817 439 — EERRS (SEQ ID NO: 0.185685948 0.095520154 3908) 152 — TN 0.185685948 0.085877547 684 — LGN 0.18563709 0.122810431 718 Y [stop] 0.185557951 0.073176523 585 L P 0.185474446 0.130833458 85 W R 0.185353654 0.134359698 931 — SWLFL (SEQ ID NO: 0.185304071 0.113870586 4178) 543 — KKIK (SEQ ID NO: 0.185304071 0.066752877 3996) 547 — PEAFEAN (SEQ ID 0.185304071 0.089391329 NO: 4088) 91 D G 0.1853036 0.092089143 766 K R 0.185284272 0.110005204 461 — SFVIE (SEQ ID NO: 0.185264915 0.156592075 4150) 950 — GNTDK (SEQ ID 0.185264915 0.154386625 NO: 3953) 825 L M 0.185209061 0.126951087 727 K M 0.185134776 0.155871835 28 M K 0.1848853 0.176098567 404 H R 0.184633168 0.163423927 394 A T 0.184555363 0.1424277 581 I F 0.184470581 0.083013305 766 K M 0.184394313 0.16735316 547 P L 0.18.4346525 0.155161861 275 F S 0.184250266 0.085183481 537 G V 0.184185986 0.146420736 873 S N 0.184149692 0.143102895 198 -I CL 0.184139991 0.106675461 639 — ERR 0.184139991 0.11669463 287 -K CL 0.184067988 0.105370778 404 H N 0.183958455 0.132891407 710 — VEQRR (SEQ ID NO: 0.183918384 0.104439918 4207) 889 S P 0.183788189 0.164091129 144 V L 0.183743996 0.065170935 165 R K 0.183736362 0.17610787 28 M V 0.183560659 0.134087452 611 A T 0.183558778 0.136945744 148 GK DR 0.183483799 0.153480995 515 A C 0.183483799 0.109594032 367 N S 0.183341948 0.159877593 868 E K 0.183187044 0.163165035 306 L Q 0.183120006 0.156397405 216 G D 0.183066489 0.119789101 728 N Y 0.183065668 0.166304554 879 N I 0.183004606 0.128653405 126 G V 0.182789208 0.179342988 35 V M 0.182763396 0.156289233 443 S N 0.182633222 0.162446869 951 N D 0.182629417 0.175906154 410 G S 0.182624091 0.128840332 233 V V 0.182567289 0.115088116 96 M L 0.182378018 0.128312349 753 — IFANLS (SEQ ID NO: 0.182269944 0.088037483 3974) 634 V A 0.182243984 0.121794563 556 Y S 0.182208476 0.102238152 972 — VWKPAV (SEQ ID 0.182135365 0.122971859 NO: 4252)[stop] 716 G D 0.182118038 0.088377906 419 E G 0.182093842 0.165354368 145 N K 0.181832601 0.074663212 652 M R 0.181725898 0.15882275 183 Y [stop] 0.181723054 0.087766244 229 S R 0.18162155 0.118611624 589 K E 0.181594685 0.120760487 304 V I 0.181591972 0.14363826 873 S C 0.181321853 0.144241543 114 P S 0.181260379 0.131437002 100 A S 0.181149523 0.170663024 413 W [stop] 0.181066052 0.139390154 166 L M 0.180963828 0.128703075 496 — IEAENS (SEQ ID 0.180890191 0.096196015 NO: 3970) 504 D V 0.180843532 0.116307526 199 H Q 0.180819165 0.098967075 675 C W 0.180770613 0.172891211 94 G S 0.180639091 0.140246364 212 E D 0.180617877 0.126552831 557 T N 0.180519556 0.15369828 753 I S 0.180492647 0.165598334 872 L V 0.180432435 0.164444609 596 — IWNDLL (SEQ ID 0.180218478 0.160627748 NO: 3984) 382 SS CL 0.180218478 0.105067529 369 AG DS 0.180218478 0.132171137 757 LS PV 0.180218478 0.120148198 674 — GCPLSRFK (SEQ ID 0.180218478 0.119094301 NO: 3938) 418 — DE 0.180218478 0.162709755 702 — RTIQAAK (SEQ ID 0.180179308 0.102882749 NO: 4145) 81 L P 0.180116381 0.137095425 939 — EYK 0.18007812 0.13192478 31 L Q 0.180015666 0.152602881 213 — QIGGN (SEQ ID NO: 0.179890016 0.080439406 4104) 379 — PY 0.179789203 0.118280148 331 F Y 0.179617168 0.14637274 540 L M 0.179584486 0.167412262 693 I V 0.179569128 0.124539552 776 T S 0.179453432 0.075575874 264 L V 0.179340275 0.144429387 547 P R 0.179333799 0.110886672 820 D E 0.179273983 0.124243775 604 E K 0.17907609 0.153006263 651 P S 0.17907291 0.16196086 382 S C 0.179061797 0.042397129 680 F Y 0.179026865 0.083849485 552 A V 0.178983921 0.137645246 693 I F 0.178916903 0.17080226 151 HT LS 0.178787645 0.11267363 190 — QRALD (SEQ ID NO: 0.178787645 0.150480322 4109) 208 — VKPLE (SEQ ID NO: 0.178787645 0.112763983 4211) 194 D V 0.178645393 0.146182868 163 H R 0.178633884 0.108142143 383 I I 0.178486259 0.158810182 156 G D 0.178426488 0.134868493 234 G E 0.178414368 0.12320748 804 Y [stop] 0.178116642 0.169884859 582 I N 0.177915368 0.151449157 655 I T 0.177824888 0.131979099 129 C Y 0.177764169 0.131217004 20 K [stop] 0.177744686 0.162022223 852 Y C 0.177655192 0.126363222 179 E Q 0.177438027 0.163530401 365 W S 0.177330558 0.12784352 245 D E 0.177288135 0.128142583 593 R G 0.177150053 0.165372274 838 T S 0.177144418 0.166381063 979 LE[stop]G VSSR (SEQ ID NO: 0.177037198 0.160568847 4248) 265 K E 0.176890073 0.124809095 440 E D 0.176868582 0.097257257 107 I M 0.176863119 0.14397231 22 A P 0.176753805 0.123959084 292 A G 0.176665583 0.159949136 803 Q [stop] 0.176624558 0.101059884 329 P S 0.176586746 0.173503743 196 Y [stop] 0.176517802 0.122355941 758 S N 0.176368261 0.089480066 298 A T 0.176357721 0.087659893 333 L V 0.176333899 0.163860363 518 W R 0.176185261 0.104632883 459 KA -V 0.176164273 0.103778218 192 AL DR 0.176164273 0.079837153 979 LE-[stop]G VSSKDLQA (SEQ ID 0.176164273 0.074531926 NO: 3671) 35 VMT ETA 0.176164273 0.104758915 767 RT SC 0.176164273 0.119651092 678 S N 0.176147348 0.146692604 817 T A 0.176123605 0.120992816 635 A G 0.176061926 0.119367224 212 E A 0.175873239 0.11085302 821 Y [stop] 0.175384143 0.118184345 447 Q R 0.175284629 0.123528707 257 N S 0.175186561 0.099304683 618 K R 0.175178956 0.153225543 217 N S 0.175170771 0.153898212 852 Y [stop] 0.175104531 0.090584521 255 K R 0.175069831 0.070668507 430 — GLS 0.175035484 0.093564105 827 — KLKK (SEQ ID NO: 0.175035484 0.069987475 4004) 796 — YLS 0.175035484 0.092544675 414 — GKVYDEAWE (SEQ 0.175035484 0.140128399 ID NO: 3948) 547 — PEAFE (SEQ ID NO: 0.175035484 0.118947618 4087) 186 — GKFGQR (SEQ ID 0.175035484 0.092907507 NO: 3946) 580 L R 0.174993228 0.092760152 422 E K 0.174900558 0.171745203 285 H Y 0.17.4862549 0.137793142 737 T I 0.174757975 0.115488534 455 W G 0.174674459 0.156270727 401 L P 0.174440338 0.064966394 953 — DKR 0.17.4181069 0.090682808 953 — DKRA (SEQ ID NO: 0.174181069 0.085814279 3890) 360 D N 0.174161173 0.117286104 520 K E 0.174117735 0.143263172 145 N D 0.174107257 0.119744646 819 — ADYD (SEQ ID NO: 0.174068679 0.17309276 3846) 561 K [stop] 0.174057181 0.086009056 761 F S 0.17403349 0.168753775 563 S P 0.173902999 0.138700996 70 L P 0.173882613 0.120818159 24 K [stop] 0.173808747 0.113872328 834 G A 0.173722333 0.117168406 167 I N 0.173700086 0.14772793 496 — IEAENSILD (SEQ ID 0.173653508 0.110162475 NO: 3972) 618 K [stop] 0.173508668 0.101750483 297 V E 0.173261294 0.132967549 426 K E 0.173245682 0.081642461 182 T K 0.173138422 0.156579716 660 G S 0.17299716 0.158169348 805 T S 0.172972548 0.12868971 458 A S 0.172827968 0.144714634 731 D V 0.172739834 0.130565896 829 K E 0.172710008 0.121812751 859 Q [stop] 0.172627299 0.130823394 305 — NL 0.172611068 0.12831984 178 — DE 0.172611068 0.108355628 652 M V 0.172566944 0.106266804 582 I M 0.172413921 0.144870464 335 E G 0.172324707 0.120749484 940 — YK 0.172247171 0.104630004 450 A D 0.172235862 0.15659478 187 K T 0.172165735 0.159986695 289 GI AV 0.172163889 0.117287191 579 NL DR 0.172163889 0.094383078 813 E G 0.172115298 0.163114025 259 K E 0.171933606 0.128545463 255 K M 0.171890748 0.139268571 675 — CP 0.171877476 0.064917248 853 Y C 0.171733581 0.087723362 631 A V 0.171731995 0.15053602 668 A V 0.171647872 0.129168631 508 F S 0.17126701 0.136692573 925 AL DR 0.17104011 0.083554381 437 — LE 0.17104041 0.06885585 853 — YN 0.17104041 0.123300185 797 — LSKILA (SEQ ID 0.17104041 0.064415402 NO: 4057) 815 — TIT 0.17104041 0.104377719 462 -FV ERL[stop] 0.17104041 0.089353273 471 — DK 0.17104041 0.0730883 418 — DEAWE (SEQ ID 0.170904662 0.126366449 NO: 3879) 213 — QIG 0.170882441 0.117196646 703 — TIQA (SEQ ID NO: 0.170763645 0.147647998 4189) 356 E A 0.170659559 0.127216719 869 L V 0.170596065 0.1158133 106 NI TV 0.170299453 0.164756763 160 V L 0.170273865 0.111449611 163 H Q 0.170101095 0.104599592 210 P T 0.170021527 0.150133417 748 QD R- 0.169874659 0.074658631 775 — YIRMED (SEQ ID 0.169874659 0.080414628 NO: 4272) 513 N I 0.169811112 0.150139289 743 — YY 0.169783049 0.088429509 467 — LKEADKD (SEQ ID 0.169783049 0.163043441 NO: 4041) 663 -I CL 0.169783049 0.106475808 803 — QYTSKT (SEQ ID 0.169772888 0.094792337 NO: 4117) 808 — TCSNCG (SEQ ID 0.169772888 0.089412307 NO: 4182) 845 K E 0.169715078 0.127028772 552 A T 0.169382091 0.146396839 476 C F 0.169278987 0.093974927 711 E D 0.169174495 0.118203075 631 A S 0.169116909 0.130583861 303 W [stop] 0.169003266 0.078930757 561 K I 0.168954178 0.166308652 157 — RC 0.168739459 0.094824256 721 K R 0.168620063 0.147491806 614 R [stop] 0.168568195 0.15863634 611 A D 0.168315642 0.157590847 78 K [stop] 0.168282214 0.125424128 917 — ETHA (SEQ ID NO: 0.168207257 0.122439321 3919) 756 NL DR 0.168207257 0.079944251 678 S G 0.168124453 0.111226188 525 K I 0.16804127 0.142310409 653 N K 0.167953422 0.124668308 37 T N 0.16794635 0.137106698 174 P S 0.167775884 0.122107474 756 — NLSR (SEQ ID NO: 0.167679572 0.073550026 4074) 168 — LLSPHK (SEQ ID 0.167679572 0.081935755 NO: 4046) 160 — VSEHERLI (SEQ ID 0.167679572 0.116191677 NO: 4219) 859 — QNVVK (SEQ ID 0.167565632 0.122604368 NO: 4107) 719 S P 0.167206156 0.083551442 712 Q R 0.167205037 0.147128575 964 F S 0.166884399 0.138397154 359 E G 0.16680448 0.139659272 191 R K 0.166577954 0.144007057 339 N D 0.166374831 0.157063101 212 E K 0.166305352 0.157035199 413 WG LS 0.166270685 0.125303472 149 — KP 0.166270685 0.076773688 284 — PHTK (SEQ ID NO: 0.166270685 0.139854804 4089) 146 D N 0.166006779 0.113823305 686 N D 0.165853975 0.141480032 492 K R 0.16571672 0.088451245 580 LI PV 0.165563978 0.079217211 661 — ENI 0.165563978 0.126675099 829 K R 0.165378823 0.103172827 608 L V 0.165024412 0.161094218 451 — ALT 0.164823895 0.158152194 581 II TV 0.164823895 0.074002626 297 — VAQI (SEQ ID NO: 0.164823895 0.107420642 4199) 783 — T 0.164823895 0.135845679 496 I V 0.164665656 0.140996169 979 LE[stop]G VSSE (SEQ ID NO: 0.164491714 0.145714149 4223) 932 — WLFL (SEQ ID NO: 0.164491714 0.083188044 4254) 637 — TFERRE (SEQ ID 0.164491714 0.152633112 NO: 4186) 325 — LKG 0.164491714 0.125129505 630 — PALE (SEQ ID NO: 0.164491714 0.073996533 4083) 343 — WWDMV (SEQ ID 0.164491714 0.076194534 NO: 4259) 642 — EV 0.164491714 0.162646605 419 — EAWER (SEQ ID 0.164491714 0.082157078 NO: 3906) 360 — DG 0.164491714 0.073133393 408 K E 0.16446662 0.067392631 48 R G 0.164301321 0.157884797 613 G D 0.164218988 0.127296459 175 — EANDE (SEQ ID NO: 0.164149182 0.111610409 3904) 671 D E 0.16.4120916 0.112217289 794 — KTYLSKT (SEQ ID 0.16411942 0.087804343 NO: 4020) 599 — DLLSLE (SEQ ID NO: 0.16411942 0.120903184 3895) 58 I- LS 0.16411942 0.094001227 826 E D 0.163807302 0.112540279 889 S [stop] 0.163771981 0.149267099 199 PRLY (SEQ ID NO: 0.163715064 0.07899198 4094) 916 FET VQA 0.163715064 0.085074401 496 — IEAENSI (SEQ ID 0.163715064 0.073631578 NO: 3971) 164 — ERLI (SEQ ID NO: 0.163715064 0.124419929 3916) 345 D G 0.16357556 0.12500461 134 Q [stop] 0.163522049 0.142382805 764 — QGKRTFM (SEQ ID 0.163440941 0.098647738 NO: 4101) 107 I T 0.163178218 0.154967966 633 FVAL (SEQ ID LWP[stop] 0.163026367 0.076347451 NO: 3807) 213 — QI 0.163026367 0.09979216 186 — GKFGQ (SEQ ID 0.163026367 0.114909103 NO: 3945) 592 G D 0.162807696 0.109433096 257 N K 0.162725471 0.091658038 473 DE YH 0.162404215 0.086992333 975 P A 0.162340126 0.074611129 833 T A 0.162275301 0.096163195 871 R S 0.162178581 0.080758991 909 — EVCLN (SEQ ID NO: 0.162125073 0.14885021 3934) 341 — VD 0.162125073 0.111287809 57 PI DS 0.162125073 0.110736083 83 VY AV 0.162125073 0.121259318 643 — VLD 0.162125073 0.148280778 561 K N 0.161973573 0.145314105 349 N K 0.161796683 0.105713204 318 E R 0.161659235 0.066441966 554 — RF 0.161611946 0.149093192 505 I F 0.161489243 0.076235653 102 P T 0.161386248 0.119400583 514 CA LS 0.16113532 0.083183292 979 — VSSKDLQ (SEQ ID 0.161025471 0.108550491 NO: 3667) 445 D Y 0.161008394 0.118993907 143 Q K 0.160693826 0.130109004 547 P S 0.160635883 0.144061844 43 R Q 0.160624353 0.132247177 317 D E 0.160609141 0.14140596 807 K [stop] 0.160484146 0.104229856 572 N S 0.160431799 0.062377966 644 LD PV 0.160242602 0.128569608 699 EK DR 0.160242602 0.092172248 850 I V 0.160226988 0.152692033 100 AQ LS 0.160110772 0.101933413 558 VI CL 0.160110772 0.10892714 270 — AN 0.160110772 0.124579798 979 LE[stop]GS-PGIK VSSKDLQASNT 0.160110772 0.049257177 (SEQ ID NO: (SEQ ID NO: 4233) 3665)[stop] 484 K-WYGD (SEQ NSSLSASF (SEQ ID 0.160110772 0.077521171 ID NO: 3821) NO: 4076) 205 NH LS 0.160110772 0.08695461 281 P C 0.160110772 0.141761431 939 E R 0.160110772 0.106121188 672 — S 0.160110772 0.105653932 894 — SLLKKRFS (SEQ ID 0.160110772 0.071577892 NO: 4166) 199 HV T[stop] 0.160110772 0.129212095 47 L Q 0.159718064 0.101565653 262 A V 0.159650297 0.156994685 788 — YEGLPS (SEQ ID 0.159522485 0.129386966 NO: 4261) 529 Y N 0.159442162 0.135286632 604 E V 0.159292857 0.097301034 284 P S 0.159001205 0.153355474 750 A D 0.158401706 0.125762435 950 G A 0.158324371 0.153957854 688 T I 0.158292674 0.119969439 29 K N 0.158279304 0.142748603 372 K R 0.158267712 0.11920003 275 F L 0.158241303 0.120299703 741 L P 0.158158865 0.120228264 430 G V 0.158115277 0.126566194 921 — AEQ 0.158108573 0.11103467 242 K E 0.158032112 0.1512035 148 GK RQ 0.158026029 0.155853601 295 — NV 0.157603522 0.100157866 876 — SVNN (SEQ ID NO: 0.157603522 0.131358152 4175) 215 G A 0.157466168 0.125711629 319 A V 0.15742503 0.144655841 222 G A 0.157400391 0.107390901 523 V D 0.157098281 0.069302906 753 — IFANLSR (SEQ ID 0.157085986 0.062378414 NO: 3975) 177 N S 0.157058654 0.117427271 461 S R 0.157014829 0.122688776 823 R T 0.156977695 0.125466793 427 K M 0.156963925 0.118535881 111 K [stop] 0.156885345 0.101390983 253 V L 0.156787797 0.082680225 91 D V 0.156758895 0.14763673 71 T I 0.156624998 0.127600056 592 — GREFIW (SEQ ID 0.156575371 0.050528735 NO: 3955) 847 — EGQIT (SEQ ID NO: 0.156575371 0.108055014 3911) 111 KL S[stop] 0.156575371 0.112953961 979 L-E[stop] VSSN (SEQ ID NO: 0.156575311 0.054922359 4243) 203 — ESNHPV (SEQ ID 0.156575371 0.141927058 NO: 3917) 230 DA LS 0.156575371 0.105363533 408 — KHGED (SEQ ID 0.156575371 0.140706352 NO: 3993) 606 — GSLKLAN (SEQ ID 0.156575371 0.154364417 NO: 3958) 166 L Q 0.156435151 0.079474192 213 Q H 0.156012357 0.091435578 447 Q E 0.155900092 0.095629939 689 H P 0.155877877 0.131928361 335 E Q 0.155876225 0.110366115 84 Y D 0.155784728 0.135489779 531 I N 0.155410746 0.152604803 103 A S 0.155352263 0.149390311 661 E V 0.155230224 0.090301063 865 — LSVELDR (SEQ ID 0.15478543 0.145114034 NO: 4060) 677 LS PV 0.15478513 0.108120931 570 E G 0.154599098 0.10691093 762 G D 0.154432235 0.117428168 177 N K 0.15431964 0.1416948 484 K N 0.154291635 0.117621744 592 GRE- DNQVG (SEQ ID 0.154254957 0.077027283 NO: 3898) 704 — IQAAK (SEQ ID NO: 0.154254957 0.108682368 3979) 285 — HTKEG (SEQ ID NO: 0.154254957 0.106587271 3966) 721 KY TV 0.154254957 0.124126134 650 — KPMNLIG (SEQ ID 0.154254957 0.151047576 NO: 4014) 717 G E 0.15414714 0.124750031 667 I V 0.154117319 0.147646705 623 — RRTRQ (SEQ ID NO: 0.153993707 0.122323206 4138) 773 R G 0.153915262 0.146586561 433 — KH 0.153881949 0.097541884 35 V G 0.153666817 0.124448628 211 L V 0.153538313 0.134546484 26 G D 0.15349539 0.149545585 279 — TLPPQ (SEQ ID NO: 0.15339361 0.125011235 4191) 664 — PAVIAL (SEQ ID 0.15339361 0.13972264 NO: 4084) 377 — LLPY (SEQ ID NO: 0.15339361 0.12480719 4044) 53 N D 0.15332875 0.117758231 140 K N 0.153228737 0.097346381 694 GE DR 0.153190779 0.097274205 741 — LLYY (SEQ ID NO: 0.153190779 0.13376095 4047) 592 — GREFI (SEQ ID NO: 0.153190779 0.103123693 3954) 684 — LGNPTHI (SEQ ID 0.153117895 0.112048537 NO: 4035) 532 — INY 0.153147895 0.072663729 311 K N 0.153086255 0.08609524 678 — SRFKD (SEQ ID NO: 0.152422378 0.09122337 4171) 969 LK PV 0.152422378 0.0541377 419 EAWERIDKKV RPGRESTRRW (SEQ 0.152422378 0.081179935 (SEQ ID NO: ID NO: 4131) 3804) 670 — TD 0.152422378 0.096788119 383 — SEE 0.152422378 0.066189551 403 — LHLE (SEQ ID NO: 0.152422378 0.132942463 4036) 389 KG TV 0.152422378 0.11037889 850 — ITYYN (SEQ ID NO: 0.152422378 0.102611165 3982) 230 — DAMGAV (SEQ ID 0.152422378 0.082337669 NO: 3874) 461 — SFVI (SEQ ID NO: 0.1524.22378 0.085894307 4149) 673 E- DR 0.1524.22378 0.059554386 257 N D 0.152411625 0.106853984 590 R G 0.152081011 0.117905973 737 T N 0.151886476 0.142783247 790 G E 0.151825437 0.098317165 831 T S 0.151806143 0.14386859 906 QE PV 0.151695593 0.100183043 99 V D 0.151565952 0.12300149 959 — ETW 0.151393972 0.086210639 520 K R 0.151365824 0.113621271 852 Y N 0.151328449 0.137543743 444 E G 0.151257656 0.118296919 147 — KGK 0.15109455 0.054833005 171 — PH 0.15109455 0.08380172 925 — ALN 0.15109455 0.138412128 539 — KLRFK (SEQ ID NO: 0.15109455 0.128926028 4008) 334 — VERQANE (SEQ ID 0.15109455 0.059721295 NO: 4208) 484 KW TG 0.15109455 0.091510022 848 G- AV 0.15109455 0.104352239 236 — VASFLT (SEQ ID 0.15109455 0.088006138 NO: 4201) 880 — DIS 0.15109455 0.085164607 296 VV DR 0.15109455 0.140218943 293 YN DS 0.15109455 0.094395956 359 ED AV 0.15109455 0.062026733 210 PL RQ 0.15109455 0.109823159 758 S- TG 0.15109455 0.105413113 232 CM LS 0.15109455 0.096388212 930 RSWLFL (SEQ ID EAGCS (SEQ ID NO: 0.15109455 0.077157167 NO: 3775) 3903)[stop] 886 KG C- 0.15109455 0.085064934 594 EF DC 0.15109455 0.055097165 140 K [stop] 0.150604639 0.124522684 979 LE[stop]GS- VSSKDI (SEQ ID NO: 0.150527572 0.113935287 4228) 979 L-E[stop]G VSSKA (SEQ R) NO: 0.150527572 0.106493096 4225) 851 T A 0.150513073 0.138771627 615 V A 0.150425208 0.101961366 359 — E 0.150399286 0.136024193 508 FSKQYN (SEQ ID 0.150399286 0.049469473 NO: 3929) 202 R- SSSLASGL (SEQ ID 0.150399286 0.07714146 NO: 4174)[stop] (SEQ ID NO: 4174) 884 — WTKGR (SEQ ID 0.150399286 0.084711675 NO: 4257) 399 — GDLLLH (SEQ ID 0.150399286 0.08514719 NO: 3939) 39 D G 0.150354378 0.13986784 891 E V 0.150263535 0.113865674 450 A P 0.150166455 0.146935336 429 E D 0.149933575 0.107236607 77 K E 0.148931072 0.079170957 259 — KRLANLKD (SEQ ID 0.148805792 0.108390156 NO: 4018) 978 [stop]L GI 0.148805792 0.119775179 386 D- AV 0.148805792 0.079572543 748 QD PV 0.148805792 0.094563395 609 KL DR 0.148805792 0.060702366 699 EK DC 0.148805792 0.122863259 279 — TLP 0.148805792 0.138832536 24 K M 0.148782741 0.14630409 798 S T 0.148583442 0.105674096 349 N S 0.148310626 0.138528822 403 — LH 0.148273333 0.102736 967 — KKLKEVW (SEQ ID 0.148059201 0.11964291 NO: 3999) 157 RC LS 0.14801524 0.133243315 493 PF TV 0.1480152.1 0.059147928 188 — FGQRALD (SEQ ID 0.14801524 0.10137508 NO: 3925) 898 KR TG 0.14801524 0.120213578 186 — GK 0.14801524 0.114746024 328 F- LS 0.14801524 0.071716609 204 — SNHPVKP (SEQ ID 0.14801524 0.094645672 NO: 4168) 314 — IG 0.14801524 0.075655093 422 ER AV 0.14801524 0.044733928 64 AN DS 0.14801524 0.108571015 855 — RY 0.14801524 0.108772293 504 D E 0.147876758 0.098656217 342 D H 0.147844774 0.140125334 86 EE DR 0.147451251 0.143531987 240 — LTKY (SEQ ID NO: 0.147451251 0.080958956 4061) 942 KY NC 0.147451251 0.116243971 47 LR C- 0.147451251 0.058888218 807 KT -C 0.147451251 0.120603495 603 LE PV 0.147451251 0.066385351 873 — SEE 0.147451251 0.078348652 15 KD R- 0.147451251 0.123855007 206 HP DS 0.147451251 0.064383902 599 DL — 0.147451251 0.079608104 979 L-E[stop]GS VSSKDP (SEQ ID 0.147451251 0.049212446 NO: 4237) 979 LE[stop]GS-PGIK VSSNDLQASNK 0.147451251 0.067765787 (SEQ ID NO: (SEQ ID NO: 4247) 3665)[stop] 448 — SK 0.147451251 0.090898875 505 I- LS 0.147451251 0.077683234 398 FG SV 0.147451251 0.073631355 512 -Y DS 0.147451251 0.05128316 345 — DMVC (SEQ ID NO: 0.147451251 0.06441585 3896) 177 ND- FTG[stop] 0.147451251 0.085413531 36 MT C- 0.147451251 0.118494367 953 D- AV 0.147451251 0.040719542 451 AL DR 0.147451251 0.096339405 631 A C 0.147319263 0.109020371 848 G A 0.147279724 0.093306967 239 F S 0.147177048 0.142500129 270 A I 0.147117218 0.13621963 352 K N 0.147067273 0.12109567 563 S I 0.147049099 0.111696976 612 N K 0.146927237 0.108594483 569 M V 0.146754771 0.119310335 940 -Y SV 0.14673352 0.076906931 794 KT NC 0.14673352 0.093083088 487 — GDLR (SEQ ID NO: 0.14673352 0.141269601 3940) 717 — GY 0.14673352 0.129086357 468 — KEAD (SEQ ID NO: 0.14673352 0.112176586 3987) 102 P L 0.146729077 0.094784801 462 F V 0.146714745 0.123539268 291 E Q 0.146533408 0.078647294 657 — IDRGEN (SEQ ID 0.146511494 0.145489762 NO: 3969) 32 L F 0.146467882 0.099225719 619 T N 0.146372017 0.145146105 355 N K 0.146341962 0.141209887 132 C S 0.146274101 0.131138669 831 T A 0.146217161 0.113775751 868 E V 0.145780526 0.143894902 231 A P 0.14576396 0.105172115 944 — QTNKT (SEQ ID NO: 0.14564914 0.125394667 4115) 236 — VASFL (SEQ ID NO: 0.14564914 0.09085897 4200) 709 — EV 0.14564914 0.119119066 865 L P 0.145527367 0.10928669 510 — KQYN (SEQ ID NO: 0.145296444 0.112653295 4015) 959 — ET 0.145296444 0.114339851 414 G V 0.1451247 0.140131131 465 E G 0.144909944 0.124547249 300 I T 0.144877384 0.129206612 215 G S 0.144824715 0.07809376 288 E G 0.144744415 0.110082872 16 D N 0.144678092 0.139073977 855 R G 0.144425593 0.123370913 617 E V 0.144206082 0.126166622 918 — THAAEQAA (SEQ ID 0.143857661 0.070236443 NO: 4188) 733 — MVRN (SEQ ID NO: 0.143791778 0.090612696 4065) 217 NS TG 0.143791778 0.113745581 657 — IARGE (SEQ ID NO: 0.143791778 0.039293361 3968) 533 N S 0.14375365 0.085993529 185 — LGKFGQRA (SEQ ID 0.14367777 0.094952199 NO: 4034) 616 — IEKTLYN (SEQ ID 0.14367777 0.110151228 NO: 3973) 668 — ALTDPE (SEQ ID 0.14367777 0.113895553 NO: 3858) 259 — KRLA (SEQ ID NO: 0.14367777 0.070148108 4017) 175 E- DR 0.14367777 0.049065425 610 — LANGRV (SEQ ID 0.14367777 0.105216814 NO: 4025) 507 — GESKQYN (SEQ ID 0.14367777 0.101689858 NO: 3943) 487 — GDL 0.14367777 0.046711447 731 DD CL 0.14367777 0.067816779 265 KD R- 0.14367777 0.130304386 386 — DRK 0.14367777 0.092432212 790 — GLPSK (SEQ ID NO: 0.14367777 0.104428158 3951) 774 QY PV 0.14367777 0.076535556 910 — VC 0.14367777 0.024273265 484 KW DR 0.14367777 0.094175463 20 — CL 0.14367777 0.08704024 847 — EGQITYYN (SEQ ID 0.14367777 0.054370233 NO: 3912) 114 P L 0.143623976 0.107371623 294 N S 0.143486731 0.084830242 473 D G 0.143465301 0.122194432 376 A I 0.1434567 0.101440197 637 T A 0.143296115 0.114711319 365 W C 0.143131818 0.093254266 559 I S 0.142993499 0.107801059 671 D S 0.142731931 0.123439168 487 — GDLRGK (SEQ ID 0.14265438 0.086040474 NO: 3941) 211 LEQIG (SEQ ID RNRSA (SEQ ID NO: 0.14265438 0.100691421 NO: 3825) 4127) 26 GP CL 0.14265438 0.067388407 421 — WE 0.14265438 0.084239003 211 — LEQI (SEQ ID NO: 0.14265438 0.118588014 4030) 767 R [stop] 0.1.41592128 0.123403074 290 I N 0.141531787 0.136370873 774 Q [stop] 0.141517184 0.125118121 341 V E 0.14127686 0.094518287 176 A S 0.140653486 0.112098857 562 K N 0.140512419 0.126501373 317 D H 0.140493859 0.124148887 941 — KKYQTN (SEQ ID 0.140217655 0.077001548 NO: 4002) 147 — KGKPHTNY (SEQ ID 0.140217655 0.060731949 NO: 3992) 979 LE[stop]GS- VSSKDV (SEQ ID 0.140217655 0.126849347 NO: 4238) 342 — D 0.140217655 0.083180031 701 — QRTIQA (SEQ ID 0.140217655 0.094973524 NO: 4113) 588 G R 0.140077599 0.123307802 248 L V 0.139838145 0.132091481 641 R G 0.139811399 0.120984089 375 E G 0.13977585 0.117490416 179 E K 0.139614148 0.122113279 285 — HTK 0.139514563 0.076217964 166 — LI 0.139514563 0.075733937 786 — LAYE (SEQ ID NO: 0.139514563 0.068877295 4028) 274 AF TV 0.139413376 0.092095094 578 — PN 0.139413376 0.112737023 775 — YTRME (SEQ ID NO: 0.13869596 0.096841774 4271) 838 TING (SEQ ID PSTA (SEQ ID NO: 0.13869596 0.135948561 NO: 3833) 4095) 75 E K 0.138622423 0.112055782 556 Y C 0.138477684 0.131330328 98 R [stop] 0.138179687 0.102036322 460 A I 0.137813435 0.108501414 111 K N 0.137723187 0.11828435 566 I F 0.137434779 0.130961132 438 — EEERRS (SEQ ID 0.137192189 0.064149715 NO: 3907) 58 I M 0.13705694 0.089110339 826 E K 0.136937076 0.066669616 955 R T 0.136388186 0.086919652 400 — DLLLH (SEQ ID NO: 0.136321349 0.064628042 3892) 163 — HERLILL (SEQ ID 0.136321349 0.117792482 NO: 3962) 950 — G 0.136321349 0.089773613 353 — LINEKKE (SEQ ID 0.136321349 0.11384298 NO: 4039) 469 — EADKDEFC (SEQ ID 0.136321349 0.136235916 NO: 3901) 298 — AQIVIW (SEQ ID 0.136321349 0.124259801 NO: 3861) 967 — KKL 0.136321349 0.087024226 834 G D 0.136317736 0.131556677 675 C S 0.135933989 0.124817499 295 N D 0.135903192 0.116385268 489 L P 0.135710175 0.113005835 316 R W 0.135665116 0.08159144 782 L P 0.135444097 0.094158481 252 K I 0.135215444 0.118419704 703 — TI 0.135116856 0.093813019 671 — DPE 0.135116856 0.117221994 763 R Q 0.135073853 0.130952104 815 T S 0.135026549 0.096980291 141 L M 0.134960075 0.098794232 789 E K 0.134893603 0.120008321 36 M L 0.13488937 0.122340012 278 I F 0.134789571 0.111040576 913 NCGFET (SEQ ID EAAVQA (SEQ ID 0.134611486 0.113195929 NO: 3827) NO: 3900) 11 -R AS 0.134611486 0.123271552 978 [stop]LE[stop]GS- YVSSKDLQA (SEQ 0.134611486 0.087096491 PG (SEQ ID NO: ID NO: 4277) 3668) 247 — ILEHQK (SEQ ID 0.134611486 0.104206673 NO: 3976) 517 I I 0.134524102 0.104605605 18 N Y 0.134422379 0.132333464 804 — YTSK (SEQ ID NO: 0.134383084 0.102298299 4273) 872 — LSEESVN (SEQ ID 0.134383084 0.104954479 NO: 4056) 743 Y H 0.134286698 0.08203884 250 H Q 0.134238241 0.111012466 268 A P 0.134027791 0.098451313 978 [stop]LE[stop]GS YVSSIDLQ (SEQ ID 0.134010909 0.133274253 PG (SEQ ID NO: NO: 4276) 3668) 664 — PA 0.134010909 0.124393367 979 LE[stop]G- VSSND (SEQ ID NO: 0.133919467 0.126494561 4244) 241 T N 0.133870518 0.110803484 153 N S 0.133623126 0.12555263 196 Y H 0.133619017 0.107174466 744 Y- LS 0.133358224 0.114892564 633 F S 0.133277029 0.122435158 619 T S 0.133139525 0.08963831 742 L P 0.133131448 0.09127341 809 C [stop] 0.133028515 0.072072201 86 E D 0.132733699 0.128073996 473 D V 0.132562245 0.055193421 358 K I 0.132508402 0.120198091 476 — C 0.132326289 0.087739647 953 DK E- 0.132326289 0.066036843 770 — MAERQY (SEQ ID 0.132326289 0.083381966 NO: 4064) 887 — GRSGEAL (SEQ ID 0.132326289 0.072961347 NO: 3957) 630 P S 0.132221835 0.08064538 290 I T 0.132066117 0.101441805 81 L Q 0.132063026 0.114766305 809 C F 0.131888449 0.093326725 497 — EAENSIL (SEQ ID 0.131863052 0.100142921 NO: 3902) 717 — GYSRK (SEQ ID NO: 0.131863052 0.112950153 3960) 386 — DRKK (SEQ ID NO: 0.131863052 0.08146183 3696) 68 KL TV 0.131863052 0.070945883 700 KQ DR 0.131863052 0.063471315 831 TAT PPP 0.131863052 0.067816715 157 — RCNVS (SEQ ID NO: 0.131863052 0.080937513 3697) 953 — DKRAEV (SEQ ID 0.131771442 0.07848717 NO: 3891) 978 [stop]L GF 0.131771442 0.061548024 979 LE[stop]G VSCK (SEQ ID NO: 0.131568591 0.101292375 4216) 855 R S 0.131540317 0.054730727 128 A T 0.13150991 0.131075942 225 G R 0.131348437 0.12857841 874 E D 0.131154993 0.12741404 54 I I 0.130796445 0.072189843 568 — PM 0.130626359 0.119168349 362 K Ps 0.130604026 0.105840846 359 E V 0.130475561 0.064946527 426 — KKVE (SEQ ID NO: 0.130424348 0.109290243 4001) 300 IV DR 0.130424348 0.08495594 893 — LS 0.130424348 0.106896252 256 KN TV 0.130424348 0.057621352 767 — RTFM (SEQ ID NO: 0.130.424348 0.06446722 4143) 324 R G 0.13036573 0.130162815 460 A P 0.129809906 0.111386576 744 Y S 0.129801283 0.120155085 297 V L 0.1296923 0.098130283 979 LE VP 0.129554025 0.068280994 595 — FIWNDLL (SEQ ID 0.129554025 0.083916268 NO: 3927) 909 F C 0.129452838 0.12013501 39 D N 0.128914064 0.121593627 263 N D 0.128846416 0.111193487 403 — LHLEKKH (SEQ ID 0.128586666 0.071668629 NO: 4038) 979 LE[stop]GS-G VSSIDLV (SEQ ID 0.128586666 0.121567211 NO: 4236) 876 — SVNNDI (SEQ ID 0.128586666 0.054233667 NO: 4176) 228 — LSDACMG (SEQ ID 0.128586666 0.126842965 NO: 4055) 701 — QRTI (SEQ ID NO: 0.128586666 0.098093616 4112) 797 — LSKTLAQYT (SEQ ID 0.128586666 0.060991971 NO: 4058) 14 VK AG 0.128586666 0.085310723 423 RI LS 0.128586666 0.084850033 583 — LP 0.128586666 0.051620503 979 LE[stop]GS-PGIK VSSNDLQASN (SEQ 0.128586666 0.102476858 (SEQ ID NO: ID NO: 4246) 3665) 979 LE[stop]GS-PGIK FSSKDLQASNK 0.128586666 0.093654912 (SEQ ID NO: (SEQ ID NO: 3933) 3665)[stop] 533 — NY 0.128586666 0.127517343 563 — SGEI (SEQ ID NO: 0.128586666 0.112169649 4154) 979 L-E[stop]GS VSSKDH (SEQ ID 0.128586666 0.096285329 NO: 4227) 755 — ANLS (SEQ ID NO: 0.12851771 0.091942401 3860) 4 61 S N 0.128271168 0.11452282 8 64 D E 0.128210448 0.108842691 84 Y C 0.128022871 0.110536014 720 — RKYA (SEQ ID NO: 0.127406426 0.102905352 4126) 416 VYDEAWE (SEQ CTMRPG- (SEQ ID 0.127406426 0.059900059 ID NO: 3840) NO: 3873) 808 — TCSN (SEQ ID NO: 0.127406426 0.082184056 4181) 791 — LPSKTY (SEQ ID 0.127406426 0.108127962 NO: 4052) 162 — EHERLI (SEQ ID NO: 0.127406426 0.099109571 3913) 549 — AFEANRFY (SEQ ID 0.127406426 0.084837264 NO: 3848) 979 LE[stop]GSPGI VSSIDLQE (SEQ ID 0.127187739 0.092227907 (SEQ ID NO: NO: 4234) 3674) 445 D E 0.127007554 0.122060316 82 H N 0.126805938 0.104486705 676 P L 0.126754121 0.080812602 951 — NTDK (SEQ ID NO: 0.126641231 0.099218396 4078) 979 LE[stop]GS-PGIK VSSKDLQASNN 0.126641231 0.095848514 (SEQ ID NO: (SEQ ID NO: 4232) 3665)[stop] 204 — SNHP (SEQ ID NO: 0.126641231 0.07625836 4167) 426 KK DR 0.126641231 0.097925475 923 QAA PV- 0.126641231 0.093158654 101 QP ET 0.126641231 0.062121806 942 K-Y NCL 0.126641231 0.088910569 826 EK AV 0.126641231 0.091897908 292 — AYNNV (SEQ ID 0.126641231 0.106376872 NO: 3871) 879 — NDISSWT (SEQ ID 0.126641231 0.078787272 NO: 4070) 181 VTYSLGKFGQ -SHTAWASSD (SEQ 0.126641231 0.089695218 (SEQ ID NO: ID NO: 4160) 3839) 137 YV DR 0.126641231 0.109693213 548 — EAFE 0.126641231 0.095888318 858 — RQNVVKDL (SEQ ID 0.126641231 0.065591267 NO: 4136) 231 A C 0.126641231 0.070173983 898 KRF NCL 0.126641231 0.049641927 789 EG AV 0.126641231 0.10544887 640 RR IG 0.126641231 0.104632778 303 — WVNLN (SEQ ID 0.126641231 0.064376538 NO: 4258) 640 R- TV 0.126641231 0.051697037 890 GE DR 0.126641231 0.058497447 513 — NCAFIWQK (SEQ ID 0.126641231 0.110534935 NO: 4069) 36 MT TV 0.126641231 0.096682191 979 — AV 0.126641231 0.031136061 607 — SLK 0.126641231 0.117782054 979 LE[stop]G FSSK (SEQ ID NO: 0.126627253 0.064240928 3931) 29 KT LS 0.126627253 0.070400509 510 KQ-Y SHLQ (SEQ ID NO: 0.126602218 0.092982894 4157) 960 — TWQ 0.12652671 0.053263565 665 — AVI 0.12652671 0.057438099 675 — C 0.12652671 0.103567194 451 — ALTDWLR (SEQ ID 0.12652671 0.081452296 NO: 3859) 805 — TSKTC (SEQ ID NO: 0.12652671 0.07786947 4195) 890 -GE VAKPLLQQ (SEQ ID 0.12652671 0.093632788 NO: 4198) 885 — TK 0.12652671 0.12280066 670 — TDPEGCP (SEQ ID 0.12652671 0.087582312 NO: 4185) 344 — WD 0.12652671 0.059781458 589 K [stop] 0.126002643 0.117169902 670 T I 0.125333365 0.115123087 843 E K 0.125307936 0.1170313 209 — KPL 0.125145098 0.058688797 256 — KNEKR (SEQ ID NO: 0.125115098 0.118773295 4009) 627 — QDEPALF (SEQ ID 0.125145098 0.11944079 NO: 4100) 637 TF S- 0.125145098 0.075022945 846 — VEGQIT (SEQ ID 0.125145098 0.095200634 NO: 4205) 112 LI PV 0.125145098 0.061303825 592 GRE- DNOV (SEQ ID NO: 0.125145098 0.061215515 3897) 273 — LAFPKIT (SEQ ID 0.125145098 0.062360109 NO: 4024) 773 — RQYT (SEQ ID NO: 0.125145098 0.098790624 4137) 274 AF DS 0.125145098 0.089301627 686 N- TV 0.125145098 0.106327975 549 — A 0.125145098 0.111251903 615 — VIE 0.125145098 0.115519537 486 Y [stop] 0.12498861 0.117668911 479 E G 0.124803485 0.119823525 225 G E 0.124549307 0.110077498 123 T N 0.123826195 0.091669684 436 K E 0.123328926 0.10928445 139 Y [stop] 0.123256307 0.11129924 831 T N 0.123113024 0.105004336 147 — KGKPHTN (SEQ ID 0.123112897 0.091739528 NO: 3991) 256 — KNE 0.1228.14147 0.106923843 179 EL A- 0.122844147 0.091584443 406 — EKKHG (SEQ ID NO: 0.122844147 0.089153499 3915) 295 — NVVAQ (SEQ ID 0.122844147 0.103819809 NO: 4080) 658 D E 0.122389699 0.080353294 206 H Q 0.122384978 008971464 689 H Q 0.122256431 0.089420446 306 LN PV 0.121921649 0.07283705 620 LY PV 0.121921649 0.084823364 910 — SG 0.121685511 0.114110877 508 — FSKQYNCA (SEQ ID 0.121235544 0.060533533 NO: 3930) 314 I F 0.120726616 0.074980055 746 VT C- 0.120516649 0.087097894 910 VC CL 0.119637812 0.085877084 621 — YNRRTR (SEQ ID 0.119637812 0.065553526 NO: 4266) 467 — LKEAD (SEQ ID NO: 0.119637812 0.109940477 4040) 827 — KL 0.119637812 0.054530509 374 — QEA 0.119637812 0.063378708 145 — NDK 0.119637812 0.051846935 979 LE[stop]GSPG ESSKIDLQ (SEQ ID 0.119637812 0.067517262 (SEQ ID NO: NO: 3932) 3668) 338 — ANE 0.119637812 0.103007188 389 KG R- 0.119637812 0.050940125 669 — L 0.119637812 0.05675251 845 — KVEGQI (SEQ ID 0.119637812 0.06612892 NO: 4021) 400 — DLLLHL (SEQ ID 0.119637812 0.07276695 NO: 3893) 757 L R 0.119502434 0.108713549 578 P L 0.119430629 0.116829607 634 VA LS 0.119372647 0.100712827 510 K- SHL 0.119372647 0.080479619 979 LE[stop]G ASSK (SEQ ID NO: 0.119372647 0.074447954 3865) 798 -S TA 0.119372647 0.036802807 653 NL DR 0.119372647 0.061028998 854 -N LS 0.119372647 0.074161693 420 A S 0.119261972 0.115184751 519 — QKD 0.119051026 0.108753459 600 LLS PV- 0.119011185 0.056536344 271 — NGLAFPK (SEQ ID 0.119011185 0.073725244 NO: 4072) 51 P L 0.118978183 0.099712186 403 — LHLEK (SEQ ID NO: 0.118963684 0.11518549 4037) 457 — RAKAS (SEQ ID NO: 0.118963684 0.088377062 4118) 776 — TRME (SEQ ID NO: 0.118963684 0.083809802 4194) 320 KPLQRL (SEQ ID SHCRD[stop] (SEQ 0.118677331 0.073630679 NO: 3817) ID NO: 4156) 685 GNPT (SEQ ID ATLH (SEQ ID NO: 0.118677331 0.086334956 NO: 3811) 3867) 178 — DELV (SEQ ID NO: 0.118677331 0.101525884 3883) 587 — FGKRQG (SEQ ID 0.118677331 0.110043529 NO: 3924) 783 — TAKLAN (SEQ ID 0.118677331 0.076704941 NO: 4179) 542 — FK 0.118677331 0.098685141 733 — MVRNTAR (SEQ ID 0.118677331 0.078476963 NO: 4066) 396 — YQFG (SEQ ID NO: 0.118677331 0.08225792 4268) 837 — TTING (SEQ ID NO: 0.118677331 0.059978646 4196) 729 L P 0.118360335 0.091091038 194 D E 0.117679069 0.090466918 582 ILP SC- 0.11732562 0.090313521 901 — SHR 0.11712133 0.108.439325 67 N D 0.116939695 0.113264127 309 W R 0.116671977 0.111491729 74 T S 0.11653877 0.0855649 838 T N 0.116394614 0.094955966 137 Y [stop] 0.116334699 0.088258455 591 Q [stop] 0.116290785 0.093561727 686 N K 0.116232458 0.062605741 445 — DAQSK (SEQ ID NO: 0.115532631 0.10378499 3875) 134 Q P 0.114967131 0.11371497 698 — KE 0.114412847 0.098843087 701 QR PV 0.114412847 0.104102361 281 — PPQ 0.114412847 0.077542482 708 K [stop] 0.113715295 0.106986973 696 SYK LQR 0.113676993 0.07036758 703 — TIQ 0.113676993 0.062517799 596 I F 0.113504467 0.107709004 160 — VSEHE (SEQ ID NO: 0.113504256 0.099167163 4217) 745 — AVTQD (SEQ ID 0.113504256 0.111375922 NO: 3869) 570 E K 0.1130503 0.100973674 368 L P 0.111983406 0.095724154 275 F Y 0.111191948 0.100665217 521 D E 0.111133748 0.10058089 562 K E 0.110566391 0.097349138 136 L Q 0.110244812 0.107286129 411 E G 0.110174632 0.097582202 381 LS PV 0.110164473 0.095898615 616 I V 0.109853606 0.094001833 843 E R 0.109803145 0.097494217 676 P H 0.109607681 0.091744681 484 KVVYG (SEQ ID NSSL (SEQ ID NO: 0.109535927 0.106819917 NO: 3820) 3763) 511 QY PV 0.109451554 0.106726398 979 LE[stop]GSP VSSKDV (SEQ ID 0.108902792 0.077647274 NO: 4239) 420 A V 0.108649806 0.097722159 53 N K 0.108567111 0.086753227 114 P A 0.108538006 0.106859466 637 — TFERREV (SEQ ID 0.108360722 0.063051456 NO: 4187) 286 TK DR 0.108360722 0.053025872 249 EH AV 0.108360722 0.095653705 67 NK DR 0.108360722 0.039884349 944 — QTNKTTG (SEQ ID 0.108360722 0.078648908 NO: 4116) 197 — SIHVIRE (SEQID 0.108360722 0.081689422 NO: 4161) 510 KQYNCA (SEQ ID SHLQNS (SEQ ID 0.108360722 0.044585998 NO: 3818) NO: 4158) 953 D C 0.108360722 0.098828046 63 RA SC 0.108360722 0.091093584 597 — WNDLL (SEQ ID 0.108360722 0.065802495 NO: 4255) 208 VK CL 0.108360722 0.044537036 468 — KEADKDE (SEQ ID 0.108360722 0.074432186 NO: 3988) 84 -Y DS 0.108360722 0.088490546 496 — IE 0.108360722 0.07371372 672 P-E SGCV (SEQ ID NO: 0.108360722 0.07159837 4153)[stop] 910 VC AV 0.108360722 0.062775349 868 EL DR 0.108360722 0.050620256 235 — AV 0.108360722 0.094955272 332 PL RQ 0.108360722 0.062876398 461 — SFVIEGLK (SEQ ID 0.108360722 0.064022496 NO: 4151) 562 KSGEI (SEQ ID SPAR- (SEQ ID NO: 0.108360722 0.067951904 NO: 3819) 4169) 556 — YTVINKK (SEQ ID 0.108360722 0.070852948 NO: 4274) 121 RLT SC- 0.108360722 0.070897115 868 EL NW 0.108360722 0.108128749 715 — AVTQ (SEQ ID NO: 0.108360722 0.088762315 3868) 513 — NCAFIW (SEQ ID 0.108360722 0.045078115 NO: 4068) 429 — EGLS (SEQ ID NO: 0.108360722 0.046808088 3910) 615 VI AV 0.108360722 0.089957198 927 — NIAR (SEQ ID NO: 0.108360722 0.096224338 4073) 56 Q V 0.108360722 0.076115958 852 YY C- 0.108360722 0.054744482 816 IT LS 0.108360722 0.074232993 210 P S 0.108088041 0.085752595 251 — QKV 0.107840626 0.092439 351 — KKLI (SEQ ID NO: 0.107840626 0.05939446 3997) 962 — QSFYRKK (SEQ ID 0.107840626 0.060903469 NO: 4114) 594 EFI DCL 0.107840626 0.078577001 600 — LLS 0.107840626 0.107212137 979 LE[stop]GS-PGIK ASSKDLQASN (SEQ 0.107840626 0.073484536 (SEQ ID NO: ID NO: 3866) 3665) 606 — GSL 0.107840626 0.104907627 604 — ETG 0.107840626 0.105428162 473 — DEFCRCE (SEQ ID 0.107840626 0.072973962 NO: 3882) 798 — SKTLAQ (SEQ ID 0.107840626 0.085530107 NO: 4163) 607 — SLKLA (SEQ ID NO: 0.107840626 0.087611083 4165) 705 Q- ET 0.107840626 0.102652999 674 — GCPLSR (SEQ ID 0.107840626 0.089241733 NO: 3937) 185 — LGKEGQR (SEQ ID 0.107840626 0.068363178 NO: 4033) 344 WD LS 0.107840626 0.066070011 274 — AF 0.101840626 0.075101467 577 D G 0.1075508 0.10472372 700 K M 0.107451835 0.099853237 641 — RE 0.106527066 0.104478931 599 — DLLS (SEQ ID NO: 0.106527066 0.100649327 3894) 564 GE DR 0.106527066 0.090487961 836 MT IC 0.106527066 0.100530022 853 — YNRYK (SEQ ID NO: 0.106527066 0.088862545 4267) 586 — AFGK (SEQ ID NO: 0.106527066 0.08642655 3849) 275 -F SV 0.106527066 0.099879454 429 — EG 0.106527066 0.066947062 612 N T 0.106459427 0.08415093 611 — ANG 0.105912094 0.09807063 563 — SGEIV (SEQ ID NO: 0.105912094 0.10402865 4155) 203 E- DR 0.10545658 0.048953383 872 — LS 0.10545658 0.08227801 291 EA -C 0.10545658 0.078263499 894 S- IG 0.10545658 0.07781616 851 -T LS 0.10545658 0.071676834 251 — OK 0.105199237 0.101057895 194 — DFYSI (SEQ ID NO: 0.105199237 0.05958457 3884) 236 — VAS 0.105199237 0.084024149 899 RF SC 0.105199237 0.046835281 215 GG CL 0.105199237 0.057087854 886 KG TV 0.105199237 0.077099458 198 -I TV 0.105199237 0.087584827 878 NN DS 0.105199237 0.079694461 76 MK IC 0.105199237 0.090203405 227 ALSDA (SEQ ID SPERR (SEQ ID NO: 0.105199237 0.101107303 NO: 3800) 4170) 134 Q-P HCL 0.105199237 0.057452451 794 K-T NCL 0.105199237 0.0553.44005 532 — INYFK (SEQ ID NO: 0.105199237 0.091675146 3978) 558 VI AV 0.105199237 0.093989814 610 — LA 0.105199237 0.085523633 82 -H DS 0.105199237 0.045790293 780 DW AV 0.105199237 0.092887336 708 — KEVEQR (SEQ ID 0.105052225 0.060231645 NO: 3990) 548 EAFE (SEQ ID RPSR (SEQ ID NO: 0.105052225 0.087924295 NO: 3803) 4132) 251 — QKVIK (SEQ ID NO: 0.105052225 0.044504449 4106) 497 EA AV 0.105052225 0.084527693 841 — GKELKVE (SEQ ID 0.105052225 0.091417746 NO: 3944) 575 F- LS 0.105052225 0.076582865 910 — VCLNC (SEQ ID NO: 0.105052225 0.090851749 4202) 570 — EVNEN (SEQ ID NO: 0.104207678 0.100821855 3921) 661 — EN 0.104134797 0.102286534 500 — NSI 0.104134797 0.058937244 420 — AWERIDK (SEQ ID 0.104134797 0.06870659 NO: 3870) 533 — NYFK (SEQ ID NO: 0.104134797 0.074535749 4082) 747 — TQD 0.104134797 0.072847901 371 — YK 0.10.4134797 0.087850723 625 TR -Q 0.104134797 0.077810682 195 — FY 0.104134797 0.074775738 464 — IE 0.103802674 0.096071807 451 A T 0.103708002 0.093659384 245 DII ETV 0.10291048 0.070762893 504 — DISG (SEQ ID NO: 0.10291048 0.066659076 3887) 323 -Q IH 0.10291048 0.071312882 638 — FERRE (SEQ ID NO: 0.10291048 0.096842919 3923) 593 — REFIWNDLL (SEQ 0.10291048 0.079136445 ID NO: 4121) 730 — ADDMVR (SEQ ID 0.10291048 0.102673345 NO: 3845) 827 KL TV 0.10291048 0.094773598 138 VY C- 0.10291048 0.091363063 310 QK DR 0.10291048 0.068590108 524 KKL RN[stop] 0.102360708 0.063041226 940 — YKKYQ (SEQ ID NO: 0.102324952 0.078047936 4263) 918 — THA 0.102324952 0.066375654 979 LE[stop]GSPG VSSNDLQ (SEQ ID 0.102324952 0.073267994 (SEQ ID NO: NO: 4245) 3668) 4 K Q. 0.101594625 0.098660596 589 — KRQGR (SEQ ID 0.101233118 0.096410486 NO: 4019) 211 — LEQIG (SEQ ID NO: 0.101233118 0.097193308 4031) 649 I N 0.101148579 0.091521137 285 — HTKEGIE (SEQ ID 0.10063092 0.059060467 NO: 3967) 347 — VCN 0.10063092 0.070834064 671 — D 0.10063092 0 070617109 103 AP DS 0.10063092 0.044259819 584 — PLA 0.10063092 0.096095285 685 GN DS 0.10063092 0.057986016 837 — TTINGKE (SEQ ID 0.10063092 0 070942034 NO: 4197) 509 — SKQY (SEQ ID NO: 0.10063092 0 078527136 4162) 914 -C LS 0.10063092 0.094652044 932 — WLF 0.10063092 0.060195605 979 LE[stop]G VSRK (SEQ ID NO: 0.10063092 0.052097814 4222) 194 — DFYSIH (SEQ ID 0.10063092 0.073983623 NO: 3885) 596 — IWND (SEQ ID NO: 0.10063092 0.075782386 3983) 32 L S 0.099998377 0.098160777 822 D E 0.099951571 0.083423411 957 F S 0.099918571 0.054364404 902 — HRPV (SEQ ID NO: 0.099764722 0.080515888 3964) 474 — EFCRC (SEQ ID NO: 0.099764722 0.089224756 3909) 242 — KYQ 0.099764722 0.054563676 342 D C 0.099764722 0.075335971 413 — WG 0.099764722 0.079591734 149 — KPHTNYF (SEQ ID 0.099764722 0.070518497 NO: 4013) 510 KQY SHL 0.099764722 0.087972807 220 — ASGPVG (SEQ ID 0.099764722 0.05025267 NO: 3863) 787 AYEG (SEQ ID PTRD (SEQ ID NO: 0.099764722 0.069079749 NO: 3801) 4097) 888 — RSGEA (SEQ ID NO: 0.099764722 0.094243718 4139) 504 — DISGFS (SEQ ID 0.099764722 0.091750112 NO: 3888) 323 QR RD 0.099764722 0.040967673 647 SN DS 0.099764722 0.071118435 740 DLLY (SEQ ID NO: SAV- 0.099753827 0.050146089 3802) 38 — A 0.099114744 0.090540757 261 LA PV 0.099083678 0.060781559 255 — KKNE (SEQ ID NO: 0.098543421 0.07624083 4000) 280 — LPPQ (SEQ ID NO: 0.098543421 0.069822078 4051) 308 LW PV 0.097993366 0.087176639 753 — IFA 0.097806547 0.045793305 205 N 1 0.097706358 0.075812724 142 E Q 0.097553503 0.074603349 717 — GYSRKYAS (SEQ ID 0.097097924 0.054767341 NO: 3961) 979 LE[stop]GSPG VSSKDLH (SEQ ID 0.097097924 0.068112769 (SEQ ID NO: NO: 4229) 3668) 527 NLYL (SEQ ID NO: TCT[stop] 0.097097924 0.089930288 3828) 230 D T 0.097097924 0.061172404 595 — FIWN (SEQ ID NO: 0.097097924 0.075559339 3926) 526 LN PV 0.097097924 0.065035268 775 — YTRM (SEQ ID NO: 0.097097924 0.054287911 4270) 607 — SL 0.097097924 0.071187897 897 -K TE 0.097097924 0.05492748 118 GN DS 0.097097924 0.083309653 425 D V 0.096834118 0.093228512 704 — IQ 0.096824625 0.053400496 207 — PVKPLE (SEQ ID 0.096824625 0.074740089 NO: 4098) 154 — YF 0.096824625 0.067984555 668 — ALTD (SEQ ID NO: 0.096824625 0.088221952 3857) 386 — DR 0.096824625 0.067625309 388 — KKGK (SEQ ID NO: 0.096824625 0.060426936 3994) 880 — DISS (SEQ ID NO: 0.096824625 0.089590245 3889) 783 — TAKLAYEG (SEQ ID 0.096824625 0.064829377 NO: 4180) 643 — VLDSSNIK (SEQ ID 0.096824625 0.089286037 NO: 4213) 157 — RCN 0.096824625 0.095145301 576 — DDPNLII (SEQ ID 0.096824625 0.040738988 NO: 3877) 296 — VVAQI (SEQ ID NO: 0.096824625 0.081486595 4250) 559 -I CL 0.096824625 0.07248553 979 LE-[stop] VSIK (SEQ ID NO: 0.096824625 0.050151323 4220) 767 — RTFMAE (SEQ ID 0.096824625 0.057097889 NO: 4144) 928 IA TV 0.096824625 0.059262285 694 — GES 0.096824625 0.04858003 190 — QRA 0.096824625 0.080026424 601 — LSLETGS (SEQ ID 0.096824625 0.078527715 NO: 4059) 150 — PH 0.096482996 0.069152449 307 — NLW 0.096482996 0.053647152 808 — TCS 0.096381808 0.086676449 687 — PTHILRI (SEQ ID 0.095815136 0.067505643 NO: 4096) 469 EAD 0.095416799 0.081758814 181 VTYS (SEQ ID SHTA (SEQ ID NO: 0.095412022 0.081952005 NO: 3838) 4159) 814 F C 0.095092296 0.090308339 389 K [stop] 0.094408724 0.074513611 663 I C 0.094255793 0.075689829 979 L I 0.092483102 0.077877212 290 I- LS 0.092.483102 0.055600721 202 R-E SSSLASGL (SEQ ID 0.092483102 0.051559995 NO: 4174)[stop] 130 S I 0.092259428 0.091849472 237 A V 0.092157582 0.073154252 550 F- LS 0.091736446 0.078399586 352 — KLI 0.091736446 0.062601185 257 — NEKRLA (SEQ ID 0.091736446 0.074344692 NO: 4071) 978 [stop]LE QVS 0.091736446 0.070305933 878 NN ET 0.091736446 0.057372719 484 -KWYGD (SEQ ID NSSLSA (SEQ ID 0.091736446 0.051261975 NO: 3821) NO: 4075) 820 — DYDRVLE (SEQ ID 0.091736446 0.087280678 NO: 3899) 415 KVY Nr- 0.091736446 0.087802292 674 GCPL (SEQ ID DAh[stop] 0.091736446 0.089744971 NO: 3808) 705 QA -C 0.091.736446 0.071260814 307 -N TD 0.091736446 0.071147866 370 G - AV 0.091736446 0.05182414 954 KRA T-V 0.091736446 0.081861067 326 KGFPS (SEQ ID RASLA (SEQ ID NO: 0.091644836 0.054125593 NO: 3815) 4119) 289 GI LS 0.091644836 0.069499341 142 -E CL 0.091644836 0.064151435 10 RR TG 0.091644836 0.090788699 193 LDFYSIFI (SEQ ID RTSTAST (SEQ ID 0.091277438 0.058446074 NO: 3823) NO: 4146) 979 LE[stop]GS-PGIK VSIKDLQASNK 0.091277438 0.055852497 (SEQ ID NO: (SEQ ID NO: 4221) 3665)[stop] 590 — RQGRE (SEQ ID 0.091277438 0.07404543 NO: 4135) 308 — LWQ 0.091277438 0.063930973 311 — KLIKIGRDEA (SEQ 0.091277438 0.090951045 ID NO: 4003) 585 — LAFGKR (SEQ ID 0.091277438 0.057801256 NO: 4023) 466 — GLKEADK (SEQ ID 0.091277438 0.064806465 NO: 3950) 414 — GK 0.089604136 0.067494445 796 — YL 0.08954136 0.077067905 872 — LSE 0.089427419 0.072631533 388 — KKGKK (SEQ ID NO: 0.089427419 0.050485092 3995) 211 LEQIGG (SEQ ID RNRSAA (SEQ ID 0.089427419 0.058037112 NO: 3826) NO: 4128) 193 LDFYSIHV (SEQ RTSTAST (SEQ ID 0.089427419 0.06189365 ID NO: 3824) NO: 4147)[stop] 769 FMAERQY (SEQ LWPRGSI (SEQ ID 0.089427419 0.048645432 ID NO: 3806) NO: 4062) 558 — VIN 0.089427419 0.08506841 973 — WKP 0.089427419 0.059845159 285 — HTKE (SEQ ID NO: 0.089427419 0.058488636 3965) 353 — LI 0.089427419 0.055053978 950 — GNTD (SEQ ID NO: 0.089427419 0.068410765 3952) 642 — EVLDS (SEQ ID NO: 0.089427352 0.04064403 3920) 586 AE FT 0.089427352 0.026351335 147 KG C- 0.089427352 0.03353623 473 — DEFCR (SEQ ID NO: 0.089427352 0.087380064 3881) 62 SR CL 0.089427352 0.085389222 946 N C 0.089127352 0.086906423 341 — VDWWD (SEQ ID 0.089427352 0.088291312 NO: 4204) 546 — KPE 0.089427352 0.070048864 979 LE[stop]G-SPGI VSSKDLQACL (SEQ 0.089062173 0.059857989 (SEQ ID NO: ID NO: 4231) 3674) 979 LE[stop]GSPG ISSKDLQ (SEQ ID 0.089062173 0.071078934 (SEQ ID NO: NO: 3980) 3668) 300 — IVIW 0.089062173 0.052509601 209 KP TV 0.089062173 0.046404323 851 -T CL 0.089062173 0.047830666 466 GL LS 0.089062173 0.060367604 202 RE- SSSL (SEQ ID NO: 0.089062173 0.059904595 4173) 291 EA DC 0.089062173 0.078319771 871 RL LS 0.089062173 0.055570451 874 EE DR 0.089062173 0.077193595 868 ELDR (SEQ ID NWT- 0.089062173 0.059312334 NO: 3805) 301 VI AV 0.089062173 0.083633904 208 — VKPLEQI (SEQ ID 0.089062173 0.046331388 NO: 4212) 305 -N TT 0.089062173 0.072049193 978 [stop]L GP 0.089062173 0.071277586 866 S- TG 0.089062173 0.056416779 628 DE LS 0.089062173 0.070268313 651 -P TA 0.089062173 0.05500823 276 — PKI 0.089062173 0.06318371 299 — V 0.089062173 0.08531757 346 — MV 0.089062173 0.060831249 742 LY PV 0.089062173 0.087665343 743 YY ET 0.089062173 0.059923968 751 ML RQ 0.089062173 0.045208162 894 -S RQ 0.089062173 0.071980752 433 KH TV 0.089062173 0.061328218 899 RF LS 0.089062173 0.083069213 582 — ILP 0.089062173 0.053169618 944 — QTN 0.089062173 0.066135158 170 SP RQ 0.089062173 0.059574685 771 — AERQY (SEQ ID NO: 0.089062173 0.079591468 3847) 808 TC DS 0.089062173 0.069853908 347 — VC 0.089062173 0.085265549 554 RF SC 0.089062173 0.05713278 419 EA LS 0.089062173 0.062902243 184 — SLGKEG (SEQ ID 0.089062173 0.066443269 NO: 4164) 417 — YDEAWE (SEQ ID 0.089062173 0.084599468 NO: 4260) 911 CL DR 0.089062173 0.07167912 735 — RNTARDLLY (SEQ 0.089062173 0.058412514 ID NO: 4130) 305 N D 0.089057834 0.075458081 886 KGR RAD 0.08869535 0.056741957 235 A P 0.088591922 0.085721293 494 — FAIEAEN (SEQ ID 0.088487772 0.046582849 NO: 3922) 957 F Y 0.088355066 0.088244344 670 — TDPEG (SEQ ID NO: 0.087352311 0.070989739 4184) 388 — KK 0.087352311 0.077174067 294 — NN 0.087352311 0.079627552 748 — QDAIMLI (SEQ ID 0.087352311 0.070738039 NO: 4099) 978 [stop]LE[stop]G SVSSK (SEQ ID NO: 0.087252372 0.078631278 4177) 979 LE[stop]GS-PGIK VSSKDLHASN (SEQ 0.087252372 0.071793737 (SEQ ID NO: ID NO: 4230) 3665) 735 — RNTARD (SEQ ID 0.087252372 0.052948743 NO: 4129) 227 — ALSDACM (SEQ ID 0.087252372 0.073258454 NO: 3856) 151 HTNYFGRCNV TPTTSADATC (SEQ 0.087252372 0.05854259 (SEQ ID NO: ID NO: 4193) 3812) 875 — ESVNND (SEQ ID 0.087252372 0.069839022 NO: 3918) 151 -H CL 0.087252372 0.072166234 517 — IWQKD (SEQ ID 0.087252372 0.059389612 NO: 3985) 294 NN ET 0.087252372 0.054113615 979 LE[stop]GS-PGIK VSSEDLQASNK 0.087252372 0.053550045 (SEQ ID NO: (SEQ ID NO: 4224) 3665)[stop] 280 LP C- 0.087252372 0.046361662 973 WK CL 0.087252372 0.043130788 859 — Q 0.087252372 0.049734005 383 — SEEDR (SEQ ID NO: 0.087252372 0.079531899 4148) 193 — LDFYSIHVT (SEQ ID 0.087252372 0.075700876 NO: 4029) 731 — DDMV (SEQ ID NO: 0.087252372 0.055852115 3876) 586 — AFG 0.087252372 0.059593552 743 — YYAVTQ (SEQ ID 0.087252372 0.074424467 NO: 3799) 90 KDP NCL 0.087252372 0.062483354 459 — KAS 0.087252372 0.077679223 319 — AKPLQRLK (SEQ ID 0.087252372 0.077741662 NO: 3853) 844 — LKVEGQI (SEQ ID 0.087252372 0.078010123 NO: 4043) 964 — FYRKK (SEQ ID NO: 0.087252372 0.061717189 3935) 510 — KQYNC (SEQ ID NO: 0.087252372 0.072460113 4016) 211 LE C- 0.087252372 0.072615166 154 — YFG 0.087252372 0.050562832 428 — V 0.087252372 0.070602271 328 — FPSFPLV (SEQ ID 0.087252372 0.050986167 NO: 3928) 334 — VER 0.087252372 0.083245674 635 — ALT 0.087252372 0.058640453 87 EF DC 0.087252372 0.084662756 763 — RQGK (SEQ ID NO: 0.087252372 0.06272177 4134) 525 — KLNL (SEQ ID NO: 0.087252372 0.087055601 4005) 482 LQK PLM 0.087252372 0.0864173 228 — LS 0.087252372 0.071648918 149 — KPHT (SEQ ID NO: 0.087252372 0.063809398 4011) 14 VKDSNTK (SEQ SRTATOR (SEQ ID 0.087252372 0.086609324 ID NO: 3837) NO: 4172) 567 VP C- 0.087252372 0.05902513 11 RR GD 0.087252372 0.07840862 979 LE[stop]G VPSK (SEQ ID NO: 0.086010969 0.05573546 4215) 671 D V 0.084756133 0.072837893 462 — FVI 0.083590457 0.068208408 619 TLYNRRTR (SEQ PCTTGEPD (SEQ ID 0.083590457 0.071170573 ID NO: 3835) NO: 4086) 337 QA PV 0.083590457 0.078536227 418 — DEAW (SEQ ID NO: 0.083590457 0.038813523 3878) 426 — KK 0.083590457 0.07413354 208 VK AV 0.083590457 0.037512118 519 — QK 0.083590457 0.082570582 122 LI D[stop] 0.083590457 0.076976074 659 RG PV 0.083590457 0.0659041 160 — VSEHERL (SEQ ID 0.083590457 0.081613302 NO: 4218) 278 IT TA 0.083590457 0.047460329 242 KY CL 0.083590457 0.045794039 518 WQ GR 0.08340916 0.072293259 513 — NCAF (SEQ ID NO: 0.08340916 0.058923148 4067) 31 L C 0.082126328 0.081561344 868 E G 0.081974564 0.070868354 681 — KDSLG (SEQ ID NO: 0.080796062 0.070617083 3986) 552 — AN 0.080796062 0.080329675 168 — LLS 0.080796062 0.076933587 418 — DEAVVERID (SEQ ID 0.080796062 0.062400841 NO: 3880) 356 — EKKED (SEQ ID NO: 0.080428937 0.076250147 3914) 275 — FP 0.080428937 0.059363481 308 — LVVQKLK (SEQ ID 0.080428937 0.078547724 NO: 4063) 15 KDSNTKK (SEQ ID RTATQRR (SEQ ID 0.080428937 0.072523813 NO: 3814) NO: 4142) 979 LE[stop]GSPGI VSSKDLQG (SEQ ID 0.080428937 0.070440346 (SEQ ID NO: NO: 4235) 3674) 425 — DKK 0.080428937 0.056582403 288 EGI RAS 0.080428937 0.054809688 849 QI R- 0.080428937 0.058314054 526 — LNLYL (SEQ ID NO 0.080428937 0.073029285 4048) 546 — KPEA (SEQ ID NO: 0.080428937 0.06983999 4010) 792 — PS 0.080428937 0.067496853 706 — AAKEVEQR (SEQ ID 0.080428937 0.075434091 NO: 3843) 710 — VEQR (SEQ ID NO: 0.080165897 0.064037522 4206) 949 -T LS 0.080165897 0.057028434 224 V C 0.080165897 0.062705318 202 — RESNH (SEQ ID NO: 0.08002463 0.069004172 4122) 380 YLS -T[stop] 0.079267535 0.078743084 617 — EKT 0.079267535 0.066283102 237 AS TA 0.079267535 0.061120875 416 VYD C-T 0.07889536 0.067603097 554 — RFYIVINKK (SEQ ID 0.078495111 0.06923226 NO: 4124) 619 TLYN (SEQ ID PC-T 0.078181072 0.043873495 NO: 3834) 904 — PV 0.077521024 0.061782081 8 KR ETG 0.075979618 0.06718831 963 — SF-YR (SEQ ID NO: 0.075979618 0.064323698 4152) 34 RV SC 0.075979618 0.063118319 369 — AGYKRQ (SEQ ID 0.075979618 0.050848396 NO: 3851) 242 KY TV 0.075979618 0.056127246 297 VAQIV (SEQ ID WPRS (SEQ ID NO: 0.075979618 0.07433917 NO: 3836) 4256)[stop] 672 -P LS 0.075979618 0.056690099 650 KP TV 0.075979618 0.062837656 454 DW AV 0.075979618 0.049282705 312 LK PV 0.075979618 0.074673373 636 LT PV 0.075651042 0.051037357 325 — LKGFP (SEQ ID NO: 0.075651042 0.068819815 4042) 669 L E 0.075651042 0.075396635 79 A V 0.074780904 0.074608034 887 — GRSGEA (SEQ ID 0.073542892 0.072424639 NO: 3956) 404 HL DR 0.073542892 0.054184233 190 Q-R HVA 0.073542892 0.04828771 811 NC DS 0.073542892 0.073088889 824 — VLEK (SEQ ID NO: 0.073542892 0.055393108 4214) 63 RA TV 0.073542892 0.069467367 350 VK AV 0.072378636 0.048322939 690 ILRI (SEQ ID NO: PEN- 0.072378636 0.05860973 3813) 384 EED D-C 0.072378636 0.064425519 487 — GDLRGKP (SEQ ID 0.072378636 0.071208648 NO: 3942) 644 L [stop] 0.072378636 0.060246346 544 KI TV 0.072378636 0.05442277 933 — LFLR (SEQ ID NO: 0.072378636 0.06374014 4032) 276 PKITLP (SEQ ID LRSPCL (SEQ ID 0.072378636 0.070970251 NO: 3829) NO: 4054) 808 — TCSNCGET (SEQ ID 0.072378636 0.065622369 NO: 4183) 978 [stop]LE[stop]GS- YVSSKDL (SEQ ID 0.072378636 0.066035046 NO: 4275) 919 HA PV 0.072378636 0.058676376 378 — LPYLSSE (SEQ ID 0.072378636 0.071574474 NO: 4053) 858 RQ LS 0.072378636 0.04290216 152 — TNYFGRCN (SEQ ID 0.072378636 0.054241402 NO: 4192) 859 — QNVVKD (SEQ ID 0.072378636 0.069366552 NO: 4108) 226 KA LS 0.071324732 0.06748566 849 — QITYYN (SEQ ID 0.071251281 0.061753986 NO: 4105) 376 — ALLP (SEQ ID NO: 0.071251281 0.046839434 3854) 660 — GEN 0.071251281 0.063597301 349 — NVKKLIN (SEQ ID 0.071251281 0.055420168 NO: 4079) 427 KVE NCL 0.071251281 0.037488341 537 GGKLRFK (SEQ ID AASCGSR (SEQ ID 0.071251281 0.047685675 NO: 3809) NO: 3844) 486 — YGDLR (SEQ ID NO: 0.071251281 0.057530117 4262) 586 — AFGKRQG (SEQ ID 0.071251281 0.055531139 NO: 3850) 850 — ITYY (SEQ ID NO: 0.071251281 0.070061657 3981) 929 — ARS 0.071251281 0.070844259 617 EK AV 0.071251281 0.056273969 977 V[stop] AV 0.071036023 0.057250091 522 — GVK 0.071036023 0.066325629 903 RP LS 0.070891186 0.042147704 689 HI P- 0.070270828 0.063050321 663 — I 0.070270828 0.06150934 649 IK RQ 0.070270828 0.060617973 258 — EK 0.070270828 0.058125711 152 TN DS 0.070270828 0.059660679 351 — KKLINE (SEQ ID NO: 0.070270828 0.061736597 3998) 763 — RQ 0.070270828 0.05541295 666 VI DS 0.070270828 0.069953364 186 GK RQ 0.066783091 0.059043838 242 — KYQDIILE (SEQ ID 0.066783091 0.058248788 NO: 4022) 190 — QRALDFYS (SEQ ID 0.066783091 0.060436783 NO: 4110) 615 VI DS 0.066783091 0.065544343 295 — NVVAQI (SEQ ID 0.066783091 0.066726619 NO: 4081) 549 AFE PTR 0.066783091 0.063271062 924 -AL PSG 0.066783091 0.057049314 979 LE[stop] VSR 0.06547263 0.059545386 284 P L 0.06489326 0.063807972 620 — LY 0.06268189 0.052769076 668 -A LS 0.06268489 0.057930418 651 — PMNL (SEQ ID NO: 0.06268489 0.054376534 4091) 723 -SK PPLL (SEQ ID NO: 0.061911903 0.057719078 4093) 788 YEG TRD 0.061911903 0.061258021 572 NE DS 0.061911903 0.059419672 943 — YQTN (SEQ ID NO: 0.061911903 0.05179175 4269) 979 LE[stop]GS-P VSSKDVQ (SEQ ID 0.061911903 0.05324798 NO: 4240) 49 KK RS 0.061911903 0.057783548 745 -A LS 0.061911903 0.055420231 262 -AN ETD 0.061911903 0.056977155 726 — AKNL (SEQ ID NO: 0.061911903 0.05965082 3852) 583 — LPLA (SEQ ID NO: 0.061911903 0.053222838 4050) 585 — LA 0.061911903 0.047677961 347 — VCNVKKLI (SEQ ID 0.061911903 0.060561898 NO: 4203) 735 RN Q- 0.061911903 0.057911259 176 AN TD 0.061911903 0.042711394 484 -KWYGDL (SEQ NSSLSASF (SEQ ID 0.061911903 0.060235262 ID NO: 3822) NO: 4077) 416 VY CT 0.061911903 0.058375882 900 FS SV 0.060850202 0.045333847 550 FE CL 0.060850202 0.050669807 169 LS -P 0.059253838 0.055169203 487 GD CL 0.058561444 0.050771143 800 — TLAQYT (SEQ ID 0.058239485 0.054115265 NO: 4190) 863 KD RI 0.058239485 0.041340026 407 KKHGE (SEQ ID RSTAR (SEQ ID NO: 0.058239485 0.049050481 NO: 3816) 4141) 593 — REFIW (SEQ ID NO: 0.058239485 0.057097188 4120) 979 LE[stop]G-SP VSSKVLQ (SEQ ID 0.050653241 0.049828056 NO: 4241) 42 ER A- 0.050653241 0.043693463 897 — KK 0.050653241 0.046680114 294 NN DS 0.049177787 0.048944158 186 GKEGQRALDFY ASSDREPWTST 0.049177787 0.048777834 (SEQ ID NO: (SEQ ID NO: 3864) 3810) 696 SYK -LQ 0.049177787 0.048584657 552 AN DS 0.049177787 0.044744659 979 LE[stop]G-SPGIK VSSKYLQASNK 0.049086177 0.048688856 (SEQ ID NO: (SEQ ID NO: 4242) 3665)[stop] (SEQ ID NO: 3665) 413 — WGKVYDEA (SEQ 0.048681821 0.046101055 ID NO: 4253) 920 — AAEQA (SEQ ID 0.048224673 0.046055533 NO: 3842) 979 LE[stop]GSPG VSSKDFQ (SEQ ID 0.047884408 0.043419619 (SEQ ID NO: NO: 4226) 3668) 423 RIDKKV (SEQ ID -NRQ 0.046868759 0.045505043 NO: 3830) 162 EH AV 0.043166861 0.040108447 741 LLY CC- 0.041101883 0.039741701 443 SEDAQS (SEQ ID RGRP (SEQ ID NO: 0.041101883 0.03770041 NO: 3831) 4125)|[stop] 767 RT TA 0.041101883 0.040956261 In Table 6, [stop] represent a stop codon, so that amino acids that follow are additional amino acids after a stop codon. (−) holds the position for the insertion shown in the adjacent “Alteration” column. Pos.: Position; Ref.: Reference; Alt.: Alternation; Med. Enrich.: Median Enrichment.

Example 5: Cleavage Activity of Selected CasX Protein Variants and Variant Protein:sgRNA Pairs

The effect of select CasX protein variants on CasX protein activity, using a reference sgRNA scaffold (SEQ ID NO: 5) and E6 and/or E7 spacers is shown in Table 7 below and FIGS. 10 and 11.

In brief, EGFP HEK293T reporter cells were seeded into 96-well plates and transfected according to the manufacturer's protocol with lipofectamine 3000 (Life Technologies) and 100-200 ng plasmid DNA encoding the variant CasX protein, P2A-puromycin fusion and the reference sgRNA. The next day cells were selected with 1.5 μg/ml puromycin for 2 days and analyzed by fluorescence-activated cell sorting 7 days after selection to allow for clearance of EGFP protein from the cells EGFP disruption via editing was traced using an Attune NxT Flow Cytometer and high-throughput autosampler.

TABLE 7 Effect of CasX Protein Variants. These mutations are relative to SEQ ID NO: 2. Normalized Editing Standard SEQ ID Activity Deviation Mutation Descriptor NO 3.56 0.479918161 L379R + C477K + A708K + [P793] + T620P 3301 3.44 0.065473567 M771A 3302 3.25 0.243066966 L379R + A708K + [P793] + D732N 3303 3.2 0.065443719 W782Q 3304 3.08 0.06581193 M771Q 3305 3.06 0.098482124 R458I + A739V 3306 2.99 0.249667198 L379R + A708K + [P793] + M771N 3307 2.98 0.226829483 L379R + A708K + [P793] + A739T 3308 2.98 0.230093698 L379R + C477K + A708K + [P793] + D489S 3309 2.95 0.225022742 L379R + C477K + A708K + [P793] + D732N 3310 2.95 0.048047426 V711K 3311 2.85 0.244869555 L379R + C477K + A708K + [P793] + Y797L 3312 2.84 0.16661152 L379R + A708K + [P793] 3313 2.82 0.219742241 L379R + C477K + A708K + [P793] + M771N 3314 2.75 0.215673641 A708K + [P793] + E386S 3315 2.71 0.10301172 L379R + C477K + A708K + [P793] 3316 2.62 0.066259269 L792D 3317 2.61 0.069056066 G791F 3318 2.56 0.138158681 A708K + [P793] + A739V 3319 2.52 0.110846334 L379R + A708K + [P793] + A739V 3320 2.5 0.070762901 C477K + A708K + [P793] 3321 2.47 0.180431811 L249I, M771N 3322 2.46 0.050035486 V747K 3323 2.42 0.14702229 L379R + C477K + A708K + [P793] + M779N 3324 2.36 0.045498608 F755M 3325 2.3 0.179759799 L379R + A708K + [P793] + G791M 3326 2.29 0.16573206 E386R + F399L + [P793] 3327 2.24 0.000278715 A708K + [P793] 3328 2.23 0.243365847 L404K 3329 2.16 0.019745961 E552A 3330 2.13 0.002238075 A708K 3331 2.08 0.316339196 M779N 3332 2.08 0.062500445 P793G 3333 2.07 0.117354932 L379R + C477K + A708K + [P793] + A739V 3334 2.03 0.057771128 L792K 3335 2.01 0.186905281 L379R + A708K + [P793] + M779N 3336 2.01 0.080358848 {circumflex over ( )}AS797 3337 1.95 0.218366091 C477H 3338 1.95 0.040076499 Y857R 3339 1.94 0.032799694 L742W 3340 1.94 0.038256856 I658V 3341 1.93 0.055533894 C477K + A708K + [P793] + A739V 3342 1.9 0.028572575 S932M 3343 1.84 0.115143156 T620P 3344 1.81 0.18802403 E385P 3345 1.81 0.049828835 A708Q 3346 1.76 0.043121298 L307K 3347 1.7 0.03352434 L379R + A708K + [P793] + D489S 3348 1.7 0.170748704 C477Q 3349 1.65 0.051918988 Q804A 3350 1.64 0.169459451 F399L 3351 1.64 0.02984323 L379R + A708K + [P793] + Y797L 3352 1.64 0.168799771 L379R + C477K + A708K + [P793] + G791M 3353 1.63 0.035361733 D733T 3354 1.63 0.062042898 P793Q 3355 1.6 0.000928887 A739V 3356 1.59 0.208295832 E386S 3357 1.58 0.00189514 F536S 3358 1.57 0.204148363 D387K 3359 1.55 0.198137682 E386N 3360 1.52 0.000291529 C477K 3361 1.51 0.00032232 C477R 3362 1.49 0.095600844 A739T 3363 1.46 0.051799824 S219R 3364 1.41 0.000272809 K416E & A708K 3365 1.4 4.65E−05 L379R 3366 1.38 0.043395969 E385K 3367 1.36 0.000269797 G695H 3368 1.35 0.02584186 L379R + C477K + A708K + [P793] + A739T 3369 1.35 0.158192737 E292R 3370 1.34 0.184524879 L792K 3371 1.31 0.064556939 K25R 3372 1.31 0.08768015 K975R 3373 1.31 0.062237773 V959M 3374 1.29 0.092916832 D489S 3375 1.29 0.137197584 K808S 3376 1.28 0.181775511 N952T 3377 1.27 0.031730102 K975Q 3378 1.25 0.030353503 S890R 3379 1.23 0.350374014 [P793] 3380 1.21 8.61E−05 A788W 3381 1.21 0.057483618 Q338R + A339E 3382 1.21 0.116491085 I7F 3383 1.21 0.061416272 QT945KI 3384 1.21 0.091585825 K682E 3385 1.19 0.000423928 E385A 3386 1.19 0.053255444 P793S 3387 1.18 0.043774095 E385Q 3388 1.18 0.124987984 D732N 3389 1.17 0.101573595 E292K 3390 1.16 0.000245107 S794R + Y797L 3391 1.15 0.160445636 G791M 3392 1.14 0.098217225 I303K 3393 1.12 0.000275601 {circumflex over ( )}AS793 3394 1.11 0.037923895 S603G 3395 1.08 6.48E−05 Y797L 3396 1.08 0.034990079 A377K 3397 1.08 0.059730153 K955R 3398 1.04 0.000376903 T886K 3399 1.03 0.036131932 Q338R + A339K 3400 1.03 0.031397109 P283Q 3401 1.01 0.000158685 D600N 3402 1.01 0.095937558 S867R 3403 1.01 0.079977243 E466H 3404 1 0.086320071 E53K 3405 0.98 0.123364563 L792E 3406 0.97 5.98E−05 Q338R 3407 0.96 0.059312097 H152D 3408 0.95 0.122246867 V254G 3409 0.94 0.072611815 TT949PP 3410 0.93 0.091846036 I279F 3411 0.93 0.031803852 L897M 3412 0.92 0.000288973 K390R 3413 0.91 0.000565042 K390R 3414 0.89 0.001316868 L792G 3415 0.89 0.000623156 A739V 3416 0.89 0.033874895 R624G 3417 0.88 0.103894502 C349E 3418 0.86 0.11267313 E498K 3419 0.85 0.079415017 R388Q 3420 0.84 0.000115651 I55F 3421 0.84 0.000383356 E712Q 3422 0.83 0.025220431 E475K 3423 0.81 0.000172705 {circumflex over ( )}AS796 3424 0.8 0.111675911 Q628E 3425 0.79 0.000114918 C479A 3426 0.79 0.001115871 Q338E 3427 0.78 0.000744903 K25Q 3428 0.76 0.000269223 {circumflex over ( )}AS795 3429 0.74 0.000437653 L481Q 3430 0.73 0.0001773 E552K 3431 0.72 0.000298273 T153I 3432 0.69 0.000273628 N880D 3433 0.68 0.000192096 G791M 3434 0.67 0.000295463 C2335 3435 0.67 0.000123996 Q367K + I425S 3436 0.67 0.000188025 L685I 3437 0.66 0.000169478 K942Q 3438 0.66 0.000374718 N47D 3439 0.66 0.138212411 V635M 3440 0.64 0.067027049 G27D 3441 0.63 0.000195863 C479L 3442 0.63 0.000439659 [P793] + P793AS 3443 0.62 0.000211625 T72S 3444 0.62 0.000217614 S270W 3445 0.61 0.00019414 A751S 3446 0.6 0.066962306 Q102R 3447 0.57 0.052391074 M734K 3448 0.53 0.000621789 {circumflex over ( )}AS795 3449 0.53 0.145184217 F189Y 3450 0.5 0.038258832 W885R 3451 0.48 0.000505099 A636D 3452 0.47 0.030480379 K416E 3453 0.46 0.428767546 R693I 3454 0.45 0.593145404 m29R 3455 0.45 0.144374311 T946P 3456 0.44 0.000253022 {circumflex over ( )}L889 3457 0.42 0.000171566 E121D 3458 0.37 0.042821047 P224K 3459 0.37 0.683382544 K767R 3460 0.36 0.026543344 E480K 3461 0.34 0.000998618 I546V 3462 0.27 0.164274898 K188E 3463 0.22 0.00106697 Y789T 3464 0.21 0.000512104 F495S 3465 0.18 0.023184407 m29E 3466 0.18 0.096249035 A238T 3467 0.17 0.000141352 d231N 3468 0.17 9.49E−05 I199F 3469 0.17 0.031218317 N737S 3470 0.16 3.87E−05 {circumflex over ( )}G661A 3471 0.12 4.08E−05 K460N 3472 0.08 0.000897639 k210R 3473 0.08 3.47E−05 G492P 3474 0.07 0.000266253 R591I 3475 0.04 6.41E−05 {circumflex over ( )}T696 3476 0.03 0.022802297 S507G + G508R 3477 0.02 0.028138538 Y723N 3478 −0.01 0.000529731 {circumflex over ( )}P696 3479 −0.01 0.038340599 g226R 3480 −0.02 0.052026759 W974G 3481 −0.04 0.000176981 {circumflex over ( )}M773 3482 −0.04 0.07902452 H435R 3483 −0.06 0.069143378 A724S 3484 −0.06 0.060317972 T704K 3485 −0.06 0.017155351 Y966N 3486 −0.08 0.036299549 H164R 3487 −0.15 0.032952207 F556I, D646A, G695D, A751S, A820P 3488 −0.17 0.04149111 D659H 3489 −0.21 0.064777446 T806V 3490 −0.24 0.001280151 Y789D 3491 −0.31 0.05332531 C479A 3492 −0.35 0.066448437 L212P 3493 [ ] indicate deletions, and ({circumflex over ( )}) indicate insertions at the specified positions of SEQ ID NO: 2. E6 and E7 spacers were used, and the data are the average of N=6 replicates. Stdev=Standard Deviation. Editing activity was normalized to that of the reference CasX protein of SEQ ID NO: 2.

Selected CasX protein variants from the DME screen and CasX protein variants comprising combinations of mutations were assayed for their ability to disrupt via cleavage and in/del formation GFP reporter expression. CasX protein variants were assayed with two targets, with 6 replicates. FIG. 10 shows the fold improvement in activity over the reference CasX protein of SEQ ID NO: 2 of select variants carrying single mutations, assayed with the reference sgRNA scaffold of SEQ ID NO: 5.

FIG. 11 shows that combining single mutations, such as those shown in FIG. 10, can produce CasX protein variants, that can improve editing efficiency by greater than two fold. The most improved CasX protein variants, which combine 3 or 4 individual mutations, exhibit activity comparable to Staphylococcus aureus Cas9 (SaCas9) which has been used in the clinic (Maeder et al. 2019, Nature Medicine 25(2):229-233).

FIGS. 12A-12B shows that CasX protein variants, when combined with select sgRNA variants, can achieve even greater improvements in editing efficiency. For example, a protein variant comprising L379K and A708K substitutions, and a P793 deletion of SEQ ID NO: 2, when combined with the truncated stem loop T10C sgRNA variant more than doubles the fraction of disrupted cells.

Example 6: CasX Protein Variants can Affect PAM Specificity

The purpose of the experiment was to demonstrate the ability of CasX variant 2 (SEQ ID NO:2), and scaffold variant 2 (SEQ ID NO:5), to edit target gene sequences at ATCN, CTCN, and TTCN PAMs in a GFP gene. ATCN, CTCN, and TTCN spacers in the GFP gene were chosen based on PAM availability without prior knowledge of potential activity.

To facilitate assessment of editing outcomes, HEK293T-GFP reporter cell line was first generated by knocking into HEK293T cells a transgene cassette that constitutively expresses GFP. The modified cells were expanded by serial passage every 3-5 days and maintained in Fibroblast (FB) medium, consisting of Dulbecco's Modified Eagle Medium (DMEM; Corning Cellgro, #10-013-CV) supplemented with 10% fetal bovine serum (FBS; Seradigm, #1500-500), and 100 Units/mL penicillin and 100 mg/mL streptomycin (100×-Pen-Strep; GIBCO #15140-122), and can additionally include sodium pyruvate (100×, Thermofisher #11360070), non-essential amino acids (100× Thermofisher #11140050), HEPES buffer (100× Thermofisher #15630080), and 2-mercaptoethanol (1000× Thermofisher #21985023). The cells were incubated at 37° C. and 5% CO2. After 1-2 weeks, GFP+ cells were bulk sorted into FB medium. The reporter lines were expanded by serial passage every 3-5 days and maintained in FB medium in an incubator at 37° C. and 5% CO2. Clonal cell lines were generated by a limiting dilution method.

HEK293T-GFP reporter cells, constructed using cell line generation methods described above were used for this experiment. Cells were seeded at 20-40k cells/well in a 96 well plate in 100 μL of FB medium and cultured in a 37° C. incubator with 5% CO2. The following day, cells were transfected at ˜75% confluence using lipofectamine 3000 and manufacturer recommended protocols. Plasmid DNA encoding CasX and guide construct (e.g., see table for sequences) were used to transfect cells at 100-400 ng/well, using 3 wells per construct as replicates. A non-targeting plasmid construct was used as a negative control. Cells were selected for successful transfection with puromycin at 0.3-3 μg/ml for 24-48 hours followed by recovery in FB medium. Edited cells were analyzed by flow cytometry 5 days after transduction. Briefly, cells were sequentially gated for live cells, single cells, and fraction of GFP-negative cells.

Results: The graph in FIG. 15 shows the results of flow cytometry analysis of Cas-mediated editing at the GFP locus in HEK293T-GFP cells 5 days post-transfection. Each data point is an average measurement of 3 replicates for an individual spacer. Reference CasX reference protein (SEQ ID NO: 2) and gRNA (SEQ ID NO: 5) RNP complexes showed a clear preference for TTC PAM (FIG. 15). This served as a baseline for CasX protein and sgRNA variants that altered specificity for the PAM sequence. FIG. 16 shows that select CasX protein variants can edit both non-canonical and canonical PAM sequences more efficiently than the reference CasX protein of SEQ ID NO: 2 when assayed with various PAM and spacer sequences in HEK293 cells. The construct with non-targeting spacer resulted in no editing (data not shown). This example demonstrates that, under the conditions of the assay, CasX with appropriate guides can edit at target sequences with ATCN, CTCN and TTCN PAMs in HEK293T-GFP reporter cells, and that improved CasX variants increase editing activity at both canonical and non-canonical PAMs.

Example 7: Reference Planctomycetes CasX RNPs are Highly Specific

Reference CasX RNP complexes were assayed for their ability to cleave target sequences with 1-4 mutations, with results shown in FIGS. 17A-17F. Reference Planctomycetes CasX RNPs were found to be highly specific and exhibited fewer off-target effects than SpyCas9 and SauCas9.

Example 8: Creation, Expression and Purification of CasX Constructs Growth and Expression

Expression constructs for the CasX of Table 8 were constructed from gene fragments (Twist Biosciences) that were codon optimized for E. coli. The assembled construct contains a TEV-cleavable, C-terminal, TwinStrep tag and was cloned into a pBR322-derivative plasmid backbone containing an ampicillin resistance gene. The sequences of Table 8 are configured as: SV40 NLS-CasX-SV40 NLS-TEV cleavage site -TwinStrep tag. Expression constructs were transformed into chemically-competent BL21*(DE3) E. coli and a starter culture was grown overnight in LB broth supplemented with carbenicillin at 37° C., 180 RPM, in UltraYield Flasks (Thomson Instrument Company). The following day, this culture was used to seed expression cultures at a 1:100 v/v ratio (starter culture:expression culture). Expression cultures were inoculated into Terrific Broth (Novagen) supplemented with carbenicillin and grown in UltraYield flasks at 37° C., 180 RPM. Once the cultures reached an OD of 0.5, they were chilled to 16° C. while shaking over 2 hours and IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, from a 1 M stock. The cultures were induced at 16° C., 180 RPM for 20 hours before being harvested by centrifugation at 4,000×g for 15 minutes, 4° C. The cell paste was weighed and resuspended in lysis buffer (50 mM HEPES-NaOH, 250 mM NaCl, 5 mM MgCl₂, 1 mM TCEP, 1 mM benzamidine-HCL, 1 mM PMSF, 0.5% CHAPS, 10% glycerol, pH 8) at a ratio of 5 mL of lysis buffer per gram of cell paste. Once resuspended, the sample was frozen at −80° C. until purification.

TABLE 8 Sequences of CasX constructs DNA Protein [SEQ ID [SEQ Construct NO] ID NO] Amino Acid Sequence WT Cas 3494 3498 MAPKKKRKVSQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPD X LRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEF sequence QKDPVGLMSRVAQPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLY of SEQ  VYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLG ID NO: 2 KFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGA fused to VASFLTKYQDIILEHGKVIKKNEKRLANLKDIASANGLAFPKITLPPQP an N HTKEGIEAYNNVVAQIVIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPL terminal VERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALLPYLS NLS SEEDRKKGKKFARYQFGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGL SKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCEL KLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLY LIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDP NLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCP LSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASK AKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQ YTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYSR VLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRL SEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAD EQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLK EVWKPAVAPKKKRKVSENLYFQGSAWSHPQFEKGGGSGGGSGGSAWSHP QFEKGRGSGC  CasX 119 3495 3499 MAPKKKRKVSQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPD LRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEF QKDPVGLMSRVAQPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLY VYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLG KFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGA VASFLTKYQDIILEHGKVIKKNEKRLANLKDIASANGLAFPKITLPPQP HTKEGIEAYNNVVAQIVIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPL VERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALLPYLS SEEDRKKGKKFARYQFGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGL SKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCEL KLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLY LIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDP NLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCP LSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASK AKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQ YTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRV LEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLS EESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHADE QAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKE VWKPAVPPAPKKKRKVSENLYFQGSAWSHPQFEKGGGSGGGSGGSAWSH  PQFEKGRGSGC  CasX 438 3496 3500 MAPKKKRKVSQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPD LRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEF QKDPVGLMSRVAQPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLY VYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLG KFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGA VASFLTKYQDIILEHGKVIKKNEKRLANLKDIASANGLAFPKITLPPQP HTKEGIEAYNNVVAQIVIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPL VERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALLPYLS SEEDRKKGKKFARYQFGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGL SKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCEL KLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLY LIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDP NLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCP LSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASK AKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQ YTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRV LEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLS EESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHADE QAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKE VWKPAVPPAPKKKRKVSENLYFQGSAWSHPQFEKGGGSGGGSGGSAWSH PQFEKGRGSGC  CasX 457 3497 3501 MAPKKKRKVSQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPD LRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEF QKDPVGLMSRVAQPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLY VYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLG KFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGA VASFLTKYQDIILEHGKVIKKNEKRLANLKDIASANGLAFPKITLPPQP HTKEGIEAYNNVVAQIVIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPL VERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALLPYLS SEEDRKKGKKFARYQFGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGL SKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCEL KLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLY LIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDP NLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQ DEPALFVALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCP LSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASK AKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQ YTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRV LEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLS EESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHADE QAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKE VWKPAVPPAPKKKRKVSENLYFQGSAWSHPQFEKGGGSGGGSGGSAWSH PQFEKGRGSGC 

Purification

Frozen samples were thawed overnight at 4° C. with gentle rocking. The viscosity of the resulting lysate was reduced by sonication and lysis was completed by homogenization in three passes at 17k PSI using an Emulsiflex C3 homogeniser (Avestin). Lysate was clarified by centrifugation at 50,000×g, 4° C., for 30 minutes and the supernatant was collected. The clarified supernatant was applied to a Heparin 6 Fast Flow column (GE Life Sciences) using an AKTA Pure 25M FPLC system (GE Life Sciences). The column was washed with 5 CV of Heparin Buffer A (50 mM HEPES-NaOH, 250 mM NaCl, 5 mM MgCl₂, 1 mM TCEP, 10% glycerol, pH 8), then with 3 CV of Heparin Buffer B (Buffer A with the NaCl concentration adjusted to 500 mM). Protein was eluted with 1.75 CV of Heparin Buffer C (Buffer A with the NaCl concentration adjusted to 1 M). The heparin eluate was applied to a StrepTactin HP column (GE Life Sciences) by AKTA FPLC. The column was washed with 10 CV of Strep Buffer (50 mM HEPES-NaOH, 500 mM NaCl, 5 mM MgCl₂, 1 mM TCEP, 10% glycerol, pH 8). Protein was eluted from the column using 2 CV of Strep Buffer with 2.5 mM Desthiobiotin added and collected in 0.8 CV fractions. CasX-containing fractions were pooled, concentrated at 4° C. using a 50 kDa cut-off spin concentrator (Millipore Sigma), and purified by size exclusion chromatography on a Superdex 200 pg column (GE Life Sciences) operated by AKTA FPLC. The column was equilibrated with SEC Buffer (25 mM sodium phosphate, 300 mM NaCl, 1 mM TCEP, 10% glycerol, pH 7.25). CasX-containing fractions that eluted at the appropriate molecular weight were pooled, concentrated at 4° C. using a 50 kDa cut-off spin concentrator, aliquoted, and snap-frozen in liquid nitrogen before being stored at −80° C.

Results

Following the growth and purification sections above, the following results were obtained.

WT CasX derived from Planctomycetes (SEQ ID NO:2): Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIGS. 24 and 26. Results from the gel filtration are shown in FIG. 25.

The average yield was 0.75 mg of purified CasX protein per liter of culture at 75% purity, as evaluated by colloidal Coomassie staining.

CasX Variant 119: Following the same expression and purification scheme for WT CasX, the following results were obtained for CasX variant 119. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIG. 28. Results from the gel filtration are shown in FIG. 27. The average yield was 11.7 mg of purified CasX protein per liter of culture at 95% purity, as evaluated by colloidal Coomassie staining.

CasX Variant 438: Following the same expression and purification scheme for WT CasX, the following results were obtained for CasX variant 438. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIGS. 29 and 31. Results from the gel filtration are shown in FIG. 30. The average yield was 13.1 mg of purified CasX protein per liter of culture at 97.5% purity, as evaluated by colloidal Coomassie staining.

CasX Variant 457: Following the same expression and purification scheme for WT CasX, the following results were obtained for CasX variant 457. Samples from throughout the purification procedure were resolved by SDS-PAGE and visualized by colloidal Coomassie staining, as shown in FIGS. 32 and 34. Results from the gel filtration are shown in FIG. 33. The average yield was 9.76 mg of purified CasX protein per liter of culture at 91.6% purity, as evaluated by colloidal Coomassie staining.

Overall, the results support that CasX variants can be produced and recovered at high levels of purity sufficient for experimental assays and evaluation.

Example 9: Design and Generation of CasX 119, 438 and 457

In order to generate the CasX 119, 438, and 457 constructs (sequences in Table 9), the codon-optimized CasX 37 construct (based on the WT CasX Stx2 construct of Example 8, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) was cloned into a mammalian expression plasmid (pStX; see FIG. 35) using standard cloning methods. To build CasX 119, the CasX 37 construct DNA was PCR amplified in two reactions using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol, using primers oIC539 and oIC88 as well as oIC87 and oIC540 respectively (see FIG. 36). To build CasX 457, the CasX 365 construct DNA was PCR amplified in four reactions using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol, using primers oIC539 and oIC212, oIC211 and oIC376, oIC375 and oIC551, and oIC550 and oIC540 respectively. To build CasX 438, the CasX 119 construct DNA was PCR amplified in four reactions using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol, using primers oIC539 and oIC689, oIC688 and oIC376, oIC375 and oIC551, and oIC550 and oIC540 respectively. The resulting PCR amplification products were then purified using Zymoclean DNA clean and concentrator (Zymo Research Cat #4014) according to the manufacturer's protocol. The pStX backbone was digested using XbaI and SpeI in order to remove the 2931 base pair fragment of DNA between the two sites in plasmid pStx34. The digested backbone fragment was purified by gel extraction from a 1% agarose gel (Gold Bio Cat #A-201-500) using Zymoclean Gel DNA Recovery Kit (Zymo Research Cat #D4002) according to the manufacturer's protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat #E2621S) following the manufacturer's protocol. Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. pStX34 includes an EF-1α promoter for the protein as well as a selection marker for both puromycin and carbenicillin. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat #M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) and following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. SaCas9 and SpyCas9 control plasmids were prepared similarly to pStX plasmids described above, with the protein and guide regions of pStX exchanged for the respective protein and guide. Targeting sequences for SaCas9 and SpyCas9 were either obtained from the literature or were rationally designed according to established methods. The expression and recovery of the CasX proteins was performed as described in Example 8, however in that Example, the DNA sequences were codon optimized for expression in E. coli.

TABLE 9 Sequences of CasX 119, 438 and 457 Construct DNA [SEQ ID NO] Protein [SEQ ID NO] CasX 119 3502 3505 CasX 457 3503 3506 CasX 438 3504 3507

Example 10: Design and Generation of CasX Constructs 278-280, 285-288, 290, 291, 293, 300, 492, and 493

In order to generate the CasX 278-280, 285-288, 290, 291, 293, 300, 492, and 493 constructs (sequences in Table 10), the N- and C-termini of the codon-optimized CasX 119 construct (based on the CasX Stx37 construct of Example 9, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) in a mammalian expression vector were manipulated to delete or add NLS sequences (sequences in Table 11). Constructs 278, 279, and 280 were manipulations of the N- and C-termini using only an SV40 NLS sequence. Construct 280 had no NLS on the N-terminus and added two SV40 NLS' on the C-terminus with a triple proline linker in between the two SV40 NLS sequences. Constructs 278, 279, and 280 were made by amplifying pStx34.119.174.NT with Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol, using primers oIC527 and oIC528, oIC730 and oIC522, and oIC730 and oIC530 for the first fragments each and using oIC529 and oIC520, oIC519 and oIC731, and oIC529 and oIC731 to create the second fragments each. These fragments were purified by gel extraction from a 1% agarose gel (Gold Bio Cat #A-201-500) using Zymoclean Gel DNA Recovery Kit (Zymo Research Cat #D4002) according to the manufacturer's protocol. The respective fragments were cloned together using Gibson assembly (New England BioLabs Cat #E2621S) following the manufacturer's protocol. Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat #M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically- or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) and following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

In order to generate constructs 285-288, 290, 291, 293, and 300, a nested PCR method was used for cloning. The backbone vector and PCR template used was construct pStx34 279.119.174.NT, having the CasX 119, guide 174, and non-targeting spacer (see Examples 8 and 9 and Tables therein for sequences). Construct 278 has the configuration SV40NLS-CasX119. Construct 279 has the configuration CasX119-SV40NLS. Construct 280 has the configuration CasX119-SV40NLS-PPP linker-SV40NLS. Construct 285 has the configuration CasX119-SV40NLS-PPP linker-SynthNLS3. Construct 286 has the configuration CasX119-SV40NLS-PPP linker-SynthNLS4. Construct 287 has the configuration CasX119-SV40NLS-PPP linker-SynthNLS5. Construct 288 has the configuration CasX119-SV40NLS-PPP linker-SynthNLS6. Constrict 290 has the configuration CasX119-SV40NLS-PPP linker-EGL-13 NLS. Construct 291 has the configuration CasX119-SV40NLS-PPP linker-c-Myc NLS. Construct 293 has the configuration CasX119-SV40NLS-PPP linker-Nucleolar RNA Helicase II NLS. Construct 300 has the configuration CasX119-SV40NLS-PPP linker-Influenza A protein NLS. Construct 492 has the configuration SV40NLS-CasX119-SV40NLS-PPP linker-SV40NLS. Construct 493 has the configuration SV40NLS-CasX119-SV40NLS-PPP linker-c-Myc NLS. Each variant had a set of three PCRs: two of which were nested, were purified by gel extraction, digested, and then ligated into the digested and purified backbone. Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into the resulting pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat #M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically- or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) and following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

In order to generate constructs 492 and 493, constructs 280 and 291 were digested using XbaI and BamHI (NEB #R0145S and NEB #R3136S) according to the manufacturer's protocol. Next, they were purified by gel extraction from a 1% agarose gel (Gold Bio Cat #A-201-500) using Zymoclean Gel DNA Recovery Kit (Zymo Research Cat #D4002) according to the manufacturer's protocol. Finally, they were ligated using T4 DNA ligase (NEB #M0202S) according to the manufacturer's protocol into the digested and purified pStx34.119.174.NT using XbaI and BamHI and Zymoclean Gel DNA Recovery Kit. Assembled products in the pStx34 were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting spacer sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting spacer sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into each pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat #M0202L) and an appropriate restriction enzyme for the respective plasmids. Golden Gate products were transformed into chemically- or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) and following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. The plasmids would be used to produce and recover CasX protein utilizing the general methodologies of Examples 8 and 9.

TABLE 10 CasX 278-280, 285-288, 290, 291, 293, 300, 492, and 493 constructs and corresponding Seq ID NOs Construct SEQ ID NO 278 3508 279 3509 280 3510 285 3511 286 3512 287 3513 288 3514 290 3515 291 3516 293 3517 300 3518 492 3519 493 3520

TABLE 11 Nuclear localization sequence list SEQ SEQ ID ID Amino Acid CasX NLS NO DNA Sequence NO Sequence 278, 279, 280, SV40 3521 CCAAAGAAGAAGCGG 352 PKKKRKV 492, 493 AAGGTC 285 SynthNL 3522 CACAAGAAGAAACAT 383 HKKKHPDASVNIFS S3 CCAGACGCATCAGTCA EFSK ACTTTAGCGAGTTCAG TAAA 286 SynthNL 3523 CAGCGCCCTGGGCCTT 394 QRPGPYDRPQRPG S4 ACGATAGGCCGCAAA PYDRP GACCCGGACCGTATGA TCGCCCT 287 SynthNL 3524 CTCAGCCCGAGTCTTA 385 LSPSLSPLLSPS S5 GTCCACTGCTTTCCCC LSPL GTCCCTGTCTCCACTG 288 SynthNL 3525 CGGGGCAAGGGTGGC 386 RGKGGKGLGIK S6 AAGGGGCTTGGCAAG GGAKRHRK GGGGGGGCAAAGAGG CACAGGAAG 290 EGL-13 3526 AGCCGCCGCAGAAAA 379 SRRRKANPTKL GCCAATCCTACAAAAC SENAKKLAKE TGTCAGAAAATGCGA VEN AAAAACTTGCTAAGG AGGTGGAAAAC 291 c-Myc 3527 CCTGCCGCAAAGCGA 354 PAAKRVKLD GTGAAATTGGAC 293 Nucleolar 3528 AAGCGGTCCTTCAGTA 375 KRSFSKAF RNA AGGCCTTT Helicase II 300 Influenza 3529 AAACGGGGAATAAAC 373 KRGINDRNIW A protein GACCGGAACTTCTGGC RGENERKTR GCGGGGAAAACGAGC GCAAAACCCGA

Example 11: Design and Generation of CasX Constructs 387, 395, 485-491, and 494

In order to generate CasX 395, CasX 485, CasX 486, CasX 487, the codon optimized CasX 119 (based on the CasX 37 construct of Example 9, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences), CasX 435, CasX 438, and CasX 484 (each based on CasX 119 construct of Example 9 encoding Planctomycetes CasX SEQ ID NO: 2, with a L379R substitution, a A708K substitution, and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) were cloned respectively into a 4 kb staging vector comprising a KanR marker, colE1 ori, and CasX with fused NLS (pStx1) using standard cloning methods. Gibson primers were designed to amplify the CasX SEQ ID NO: 1 Helical I domain from amino acid 192-331 in its own vector to replace this corresponding region (aa 193-332) on CasX 119, CasX 435, CasX 438, and CasX 484 in pStx1 respectively. The Helical I domain from CasX SEQ ID NO: 1 was amplified with primers oIC768 and oIC784 using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol. The destination vector containing the desired CasX variant was amplified with primers oIC765 and oIC764 using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol. The two fragments were purified by gel extraction from a 1% agarose gel (Gold Bio Cat #A-201-500) using Zymoclean Gel DNA Recovery Kit (Zymo Research Cat #D4002) according to the manufacturer's protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat #E262IS) following the manufacturer's protocol. Assembled products in the pStx1 staging vector were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing kanamycin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Correct clones were then cut and pasted into a mammalian expression plasmid (pStX: see FIG. 36) using standard cloning methods. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting spacer sequences that target the gene of interest were designed based on CasX PAM locations. Targeting spacer sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat #M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

In order to generate CasX 488, CasX 489, CasX 490, and CasX 491 (sequences in Table 12), the codon optimized CasX 119 (based on the CasX 37 construct of Example 9, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences), CasX 435, CasX 438, and CasX 484 (each based on CasX119 construct of Example 9 encoding Planctomyces CasX SEQ ID NO: 2, with a L379R substitution, a A708K substitution, and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) were cloned respectively into a 4 kb staging vector that was made up of a KanR marker, colE1 ori, and STX with fused NLS (pStx1) using standard cloning methods. Gibson primers were designed to amplify the CasX Stx1 NTSB domain from amino acid 101-191 and Helical I domain from amino acid 192-331 in its own vector to replace this similar region (aa 103-332) on CasX 119, CasX 435, CasX 438, and CasX 484 in pStx1 respectively. The NTSB and Helical I domain from CasX SEQ ID NO: 1 were amplified with primers oIC766 and oIC784 using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol. The destination vector containing the desired CasX variant was amplified with primers oIC762 and oIC765 using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol. The two fragments were purified by gel extraction from a 1% agarose gel (Gold Bio Cat #A-201-500) using Zymoclean Gel DNA Recovery Kit (Zymo Research Cat #D4002) according to the manufacturer's protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat #E2621S) following the manufacturer's protocol. Assembled products in the pStx1 staging vector were transformed into chemically-competent Turbo Competent s cob bacterial cells, plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing kanamycin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Correct clones were then cut and pasted into a mammalian expression plasmid (pStX; see FIG. 36) using standard cloning methods. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting spacer sequences that target the gene of interest were designed based on CasX PAM locations. Targeting spacer sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat #M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) and following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.

In order to generate CasX 387 and CasX 494 (sequences in Table 12), the codon optimized CasX 119 (based on the CasX 37 construct of Example 9, encoding Planctomycetes CasX SEQ ID NO: 2, with a A708K substitution and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) and CasX 484 (based on CasX 119 construct of Example 9 encoding Planctomycetes CasX SEQ ID NO: 2, with a L379R substitution, a A708K substitution, and a [P793] deletion with fused NLS, and linked guide and non-targeting sequences) were cloned respectively into a 4 kb staging vector that was made up of a KanR marker, colE1 ori, and STX with fused NLS (pStx1) using standard cloning methods. Gibson primers were designed to amplify the CasX Stx1 NTSB domain from amino acid 101-191 in its own vector to replace this similar region (aa 103-192) on CasX 119 and CasX 484 in pStx1 respectively. The NTSB domain from CasX Stx1 was amplified with primers oIC766 and oIC767 using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol. The destination vector containing the desired CasX variant was amplified with primers oIC763 and oIC762 using Q5 DNA polymerase (New England BioLabs Cat #M0491L) according to the manufacturer's protocol. The two fragments were purified by gel extraction from a 1% agarose gel (Gold Bio Cat #A-201-500) using Zymoclean Gel DNA Recovery Kit (Zymo Research Cat #D4002) according to the manufacturer's protocol. The insert and backbone fragments were then pieced together using Gibson assembly (New England BioLabs Cat #E2621S) following the manufacturer's protocol. Assembled products in the pStx1 staging vector were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing kanamycin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Correct clones were then cut and pasted into a mammalian expression plasmid (pStX; see FIG. 36) using standard cloning methods. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Sequences encoding the targeting sequences that target the gene of interest were designed based on CasX PAM locations. Targeting sequence DNA was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence and the reverse complement of this sequence. These two oligos were annealed together and cloned into pStX individually or in bulk by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat #M0202L) and an appropriate restriction enzyme for the plasmid. Golden Gate products were transformed into chemically or electro-competent cells such as NEB Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) and following the manufacturer's protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. Sequences of the resulting constructs are listed in Table 12.

TABLE 12 CasX 395 and 485-491 constructs and corresponding SEQ ID NOs Construct DNA [SEQ ID NO] Protein [SEQ ID NO] CasX 387 3530 3540 CasX 395 3531 3541 CasX 485 3532 3542 CasX 486 3533 3543 CasX 487 3534 3544 CasX 488 3535 3545 CasX 489 3536 3546 CasX 490 3537 3547 CasX 491 3538 3548 CasX 494 3539 3549

Example 12: Generation of RNA Guides

For the generation of RNA single guides and spacers, templates for in vitro transcription were generated by performing PCR with Q5 polymerase (NEB M0491) according to the recommended protocol, with template oligos for each backbone and amplification primers with the T7 promoter and the spacer sequence. The DNA primer sequences for the T7 promoter, guide and spacer for guides and spacers are presented in Table 13, below. The template oligos, labeled “backbone fwd” and “backbone rev” for each scaffold, were included at a final concentration of 20 nM each, and the amplification primers (T7 promoter and the unique spacer primer) were included at a final concentration of 1 uM each. The sg2, sg32, sg64, and sg174 guides correspond to SEQ ID NOS: 5, 2104, 2106, and 2238, respectively, with the exception that sg2, sg32, and sg64 were modified with an additional 5′ G to increase transcription efficiency (compare sequences in Table 13 to Table 2). The 7.37 spacer targets beta2-microglobulin (B2M). Following PCR amplification, templates were cleaned and isolated by phenol-chloroform-isoamyl alcohol extraction followed by ethanol precipitation.

In vitro transcriptions were carried out in buffer containing 50 mM Tris pH 8.0, 30 mM MgCl₂, 0.01% Triton X-100, 2 mM spermidine, 20 mM DTT, 5 mM NTPs, 0.5 μM template, and 100 μg/mL T7 RNA polymerase. Reactions were incubated at 37° C. overnight. 20 units of DNase I (Promega #M6101)) were added per 1 mL of transcription volume and incubated for one hour. RNA products were purified via denaturing PAGE, ethanol precipitated, and resuspended in 1× phosphate buffered saline. To fold the sgRNAs, samples were heated to 70° C. for 5 min and then cooled to room temperature. The reactions were supplemented to 1 mM final MgCl₂ concentration, heated to 50° C. for 5 min and then cooled to room temperature. Final RNA guide products were stored at −80° C.

TABLE 13 Sequences for generation of guide RNA Primer Sequence (SEQ RNA Product Primer ID NO) (SEQ ID NO) RNA product T7 promoter primer 3550 NA Used for all sg2 backbone fwd 3551 3563 GGUACUGGCGCUUUUAUCUCAUUACUU sg2 backbone rev 3552 UGAGAGCCAUCACCAGCGACUAUGUCG sg2.7.37 spacer 3553 UAUGGGUAAAGCGCUUAUUUAUCGGAG primer AGAAAUCCGAUAAAUAAGAAGCAUCAA AGGGCCGAGAUGUCUCGCUCCG sg32 backbone fwd 3554 3564 GGUACUGGCGCUUUUAUCUCAUUACUU sg32 backbone rev 3555 UGAGAGCCAUCACCAGCGACUAUGUCG sG32.7.37 spacer 3556 UAUGGGUAAAGCGCCCUCUUCGGAGGG primer AAGCAUCAAAGGGCCGAGAUGUCUCG sg64 backbone fwd 3557 3565 GGUACUGGCGCCUUUAUCUCAUUACUU sg64 backbone rev 3558 UGAGAGCCAUCACCAGCGACUAUGUCG sg64.7.37 spacer 3559 UAUGGGUAAAGCGCUUACGGACUUCGG primer UCCGUAAGAAGCAUCAAAGGGCCGAGA UGUCUCGCUCCG sg174 backbone fwd 3560 3566 ACUGGCGCUUUUAUCUgAUUACUUUGA sg174 backbone rev 3561 GAGCCAUCACCAGCGACUAUGUCGUAg sg174.7.37 spacer 3562 UGGGUAAAGCUCCCUCUUCGGAGGGAG primer CAUCAAAGGGCCGAGAUGUCUCGCUCC G

Example 13: RNP Assembly

Purified wild-type and RNP of CasX and single guide RNA (sgRNA) were either prepared immediately before experiments or prepared and snap-frozen in liquid nitrogen and stored at −80° C. for later use. To prepare the RNP complexes, the CasX protein was incubated with sgRNA at 1:1.2 molar ratio. Briefly, sgRNA was added to Buffer #1 (25 mM NaPi, 150 mM NaCl, 200 mM trehalose, 1 mM MgCl₂), then the CasX was added to the sgRNA solution, slowly with swirling, and incubated at 37° C. for 10 min to form RNP complexes. RNP complexes were filtered before use through a 0.22 μm Costar 8160 filters that were pre-wet with 200 μl Buffer #1. If needed, the RNP sample was concentrated with a 0.5 ml Ultra 100-Kd cutoff filter. (Millipore part #UFC510096), until the desired volume was obtained. Formation of competent RNP was assessed as described in Example 19.

Example 14: Assessing Binding Affinity to the Guide RNA

Purified wild-type and improved CasX will be incubated with synthetic single-guide RNA containing a 3′ Cy7.5 moiety in low-salt buffer containing magnesium chloride as well as heparin to prevent non-specific binding and aggregation. The sgRNA will be maintained at a concentration of 10 pM, while the protein will be titrated from 1 μM to 100 μM in separate binding reactions. After allowing the reaction to come to equilibrium, the samples will be run through a vacuum manifold filter-binding assay with a nitrocellulose membrane and a positively charged nylon membrane, which bind protein and nucleic acid, respectively. The membranes will be imaged to identify guide RNA, and the fraction of bound vs unbound RNA will be determined by the amount of fluorescence on the nitrocellulose vs nylon membrane for each protein concentration to calculate the dissociation constant of the protein-sgRNA complex. The experiment will also be carried out with improved variants of the sgRNA to determine if these mutations also affect the affinity of the guide for the wild-type and mutant proteins. We will also perform electromobility shift assays to qualitatively compare to the filter-binding assay and confirm that soluble binding, rather than aggregation, is the primary contributor to protein-RNA association.

Example 15: Assessing Binding Affinity to the Target DNA

Purified wild-type and improved CasX will be complexed with single-guide RNA bearing a targeting sequence complementary to the target nucleic acid. The RNP complex will be incubated with double-stranded target DNA containing a PAM and the appropriate target nucleic acid sequence with a 5′ Cy7.5 label on the target strand in low-salt buffer containing magnesium chloride as well as heparin to prevent non-specific binding and aggregation. The target DNA will be maintained at a concentration of 1 nM, while the RNP will be titrated from 1 pM to 100 μM in separate binding reactions. After allowing the reaction to come to equilibrium, the samples will be un on a native 5% polyacrylamide gel to separate bound and unbound target DNA. The gel will be imaged to identify mobility shifts of the target DNA, and the fraction of bound vs unbound DNA will be calculated for each protein concentration to determine the dissociation constant of the RNP-target DNA ternary complex.

Example 16: Assessing Differential PAM Recognition In Vitro

Purified wild-type and engineered CasX variants will be complexed with single-guide RNA bearing a fixed targeting sequence. The RNP complexes will be added to buffer containing MgCl2 at a final concentration of 100 nM and incubated with 5′ Cy7.5-labeled double-stranded target DNA at a concentration of 10 nM. Separate reactions will be carried out with different DNA substrates containing different PAMs adjacent to the target nucleic acid sequence. Aliquots of the reactions will be taken at fixed time points and quenched by the addition of an equal volume of 50 mM EDTA and 95% formamide. The samples will be run on a denaturing polyacrylamide gel to separate cleaved and uncleaved DNA substrates. The results will be visualized and the rate of cleavage of the non-canonical PAMs by the CasX variants will be determined.

Example 17: Assessing Nuclease Activity for Double-Strand Cleavage

Purified wild-type and engineered CasX variants will be complexed with single-guide RNA bearing a fixed PM22 targeting sequence. The RNP complexes will be added to buffer containing MgCl2 at a final concentration of 100 nM and incubated with double-stranded target DNA with a 5′ Cy7.5 label on either the target or non-target strand at a concentration of 10 nM. Aliquots of the reactions will be taken at fixed time points and quenched by the addition of an equal volume of 50 mM EDTA and 95% formamide. The samples will be run on a denaturing polyacrylamide gel to separate cleaved and uncleaved DNA substrates. The results will be visualized and the cleavage rates of the target and non-target strands by the wild-type and engineered variants will be determined. To more clearly differentiate between changes to target binding vs the rate of catalysis of the nucleolytic reaction itself, the protein concentration will be titrated over a range from 10 nM to 1 uM and cleavage rates will be determined at each concentration to generate a pseudo-Michaelis-Menten fit and determine the kcat* and KM*. Changes to KM* are indicative of altered binding, while changes to kcat* are indicative of altered catalysis.

Example 18: Assessing Target Strand Loading for Cleavage

Purified wild-type and engineered CasX 119 will be complexed with single-guide RNA bearing a fixed PM22 targeting sequence. The RNP complexes will be added to buffer containing MgCl₂ at a final concentration of 100 nM and incubated with double-stranded target DNA with a 5′ Cy7.5 label on the target strand and a 5′ Cy5 label on the non-target strand at a concentration of 10 nM. Aliquots of the reactions will be taken at fixed time points and quenched by the addition of an equal volume of 50 mM EDTA and 95% formamide. The samples will be run on a denaturing polyacrylamide gel to separate cleaved and uncleaved DNA substrates. The results will be visualized and the cleavage rates of both strands by the variants will be determined. Changes to the rate of target strand cleavage but not non-target strand cleavage would be indicative of improvements to the loading of the target strand in the active site for cleavage. This activity could be further isolated by repeating the assay with a dsDNA substrate that has a gap on the non-target strand, mimicking a pre-cleaved substrate. Improved cleavage of the non-target strand in this context would give further evidence that the loading and cleavage of the target strand, rather than an upstream step, has been improved.

Example 19: CasX:gNA In Vitro Cleavage Assays 1. Determining Cleavage-Competent Fraction

The ability of CasX variants to form active RNP compared to reference CasX was determined using an in vitro cleavage assay. The beta-2 microglobulin (B2M) 7.37 target for the cleavage assay was created as follows. DNA oligos with the sequence TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (non-target strand, NTS; SEQ ID NO: 3567) and TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (target strand, TS; SEQ ID NO: 3568) were purchased with 5′ fluorescent labels (LI-COR IRDye 700 and 800, respectively). dsDNA targets were formed by mixing the oligos in a 1:1 ratio in 1× cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl₂), heating to 95° C. for 10 minutes, and allowing the solution to cool to room temperature.

CasX RNPs were reconstituted with the indicated CasX and guides (see graphs) at a final concentration of 1 μM with 1.5-fold excess of the indicated guide in 1× cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl₂) at 37° C. for 10 min before being moved to ice until ready to use. The 7.37 target was used, along with sgRNAs having spacers complementary to the 7.37 target.

Cleavage reactions were prepared with final RNP concentrations of 100 nM and a final target concentration of 100 nM. Reactions were carried out at 37° C. and initiated by the addition of the 7.37 target DNA. Aliquots were taken at 5, 10, 30, 60, and 120 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C. for 10 minutes and run on a 10% urea-PAGE gel. The gels were imaged with a LI-COR Odyssey CLx and quantified using the LI-COR Image Studio software. The resulting data were plotted and analyzed using Prism. We assumed that CasX acts essentially as a single-turnover enzyme under the assayed conditions, as indicated by the observation that sub-stoichiometric amounts of enzyme fail to cleave a greater-than-stoichiometric amount of target even under extended time-scales and instead approach a plateau that scales with the amount of enzyme present. Thus, the fraction of target cleaved over long time-scales by an equimolar amount of RNP is indicative of what fraction of the RNP is properly formed and active for cleavage. The cleavage traces were fit with a biphasic rate model, as the cleavage reaction clearly deviates from monophasic under this concentration regime, and the plateau was determined for each of three independent replicates. The mean and standard deviation were calculated to determine the active fraction (Table 14). The graphs are shown in FIG. 37.

Apparent active (competent) fractions were determined for RNPs formed for CasX2+guide 174+7.37 spacer, CasX119+guide 174+7.37 spacer, and CasX459+guide 174+7.37 spacer. The determined active fractions are shown in Table 14. Both CasX variants had higher active fractions than the wild-type CasX2, indicating that the engineered CasX variants form significantly more active and stable RNP with the identical guide under tested conditions compared to wild-type CasX. This may be due to an increased affinity for the sgRNA, increased stability or solubility in the presence of sgRNA, or greater stability of a cleavage-competent conformation of the engineered CasX:sgRNA complex. An increase in solubility of the RNP was indicated by a notable decrease in the observed precipitate formed when CasX457 was added to the sgRNA compared to CasX2. Cleavage-competent fractions were also determined for CasX2.2.7.37, CasX2.32.7.37, CasX2.64.7.37, and CasX2.174.7.37 to be 16±3%, 13±3%, 5±2%, and 22±5%, as shown in FIG. 38.

The data indicate that both CasX variants and sgRNA variants are able to form a higher degree of active RNP with guide RNA compare to wild-type CasX and wild-type sgRNA.

2. In Vitro Cleavage Assays—Determining k_(cleave) for CasX Variants Compared to Wild-Type Reference CasX

The apparent cleavage rates of CasX variants 119 and 457 compared to wild-type reference CasX were determined using an n vitro fluorescent assay for cleavage of the target 7.37.

CasX RNPs were reconstituted with the indicated CasX (see FIG. 39) at a final concentration of 1 μM with 1.5-fold excess of the indicated guide in 1× cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl₂) at 37° C. for 10 min before being moved to ice until ready to use. Cleavage reactions were set up with a final RNP concentration of 200 nM and a final target concentration of 10 nM. Reactions were carried out at 37° C. and initiated by the addition of the target DNA. Aliquots were taken at 0.25, 0.5, 1, 2, 5, and 10 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C. for 10 minutes and run on a 10% urea-PAGE gel. The gels were imaged with a LI-COR Odyssey CLx and quantified using the LI-COR Image Studio software. The resulting data were plotted and analyzed using Prism, and the apparent first-order rate constant of non-target strand cleavage (k_(cleave)) was determined for each CasX:sgRNA combination replicate individually. The mean and standard deviation of three replicates with independent fits are presented in Table 14, and the cleavage traces are shown in FIG. 38.

Apparent cleavage rate constants were determined for wild-type CasX2, and CasX variants 19 and 457 with guide 174 and spacer 7.37 utilized in each assay. Under the assayed conditions, the k_(cleave) of CasX2, CasX119, and CasX457 were 0.51±0.01 min⁻¹, 6.29±2.11 min⁻¹ and 3.01±0.90 min⁻¹ (mean±SD), respectively (see Table 14 and FIG. 39). Both CasX variants had improved cleavage rates relative to the wild-type CasX2, though notably CasX119 has a higher cleavage rate under tested conditions than CasX457. As demonstrated by the active fraction determination, however, CasX457 more efficiently forms stable and active RNP complexes, allowing different variants to be used depending on whether the rate of cutting or the amount of active holoenzyme is more important for the desired outcome.

The data indicate that the CasX variants have a higher level of activity, with K_(cleave) rates approximately 5 to 10-fold higher compared to wild-type CasX2.

3. In Vitro Cleavage Assays: Comparison of Guide Variants to Wild-Type Guides

Cleavage assays were also performed with wild-type reference CasX2 and reference guide 2 compared to guide variants 32, 64, and 174 to determine whether the variants improved cleavage. The experiments were performed as described above. As many of the resulting RNPs did not approach full cleavage of the target in the time tested, we determined initial reaction velocities (V₀) rather than first-order rate constants. The first two timepoints (15 and 30 seconds) were fit with a line for each CasX:sgRNA combination and replicate. The mean and standard deviation of the slope for three replicates were determined.

Under the assayed conditions, the V₀ for CasX2 with guides 2, 32, 64, and 174 were 20.4±1.4 nM/min, 18.4±2.4 nM/min, 7.8±1.8 nM/min, and 49.3±1.4 nM/min (see Table 14 and FIG. 40). Guide 174 showed substantial improvement in the cleavage rate of the resulting RNP (˜2.5-fold relative to 2, see FIG. 41), while guides 32 and 64 performed similar to or worse than guide 2. Notably, guide 64 supports a cleavage rate lower than that of guide 2 but performs much better in vivo (data not shown). Some of the sequence alterations to generate guide 64 likely improve in vivo transcription at the cost of a nucleotide involved in triplex formation. Improved expression of guide 64 likely explains its improved activity in vivo, while its reduced stability may lead to improper folding in vitro.

TABLE 14 Results of cleavage and RNP formation assays RNP Construct k_(cleave)* Initial velocity* Competent fraction 2.2.7.37 20.4 ± 1.4 nM/min 16 ± 3% 2.32.7.37 18.4 ± 2.4 nM/min 13 ± 3% 2.64.7.37  7.8 ± 1.8 nM/min  5 ± 2% 2.174.7.37 0.51 ± 0.01 min⁻¹ 49.3 ± 1.4 nM/min 22 ± 5% 119.174.7.37 6.29 ± 2.11 min⁻¹ 35 ± 6% 457.174.7.37 3.01 ± 0.90 min⁻¹ 53 ± 7% *Mean and standard deviation

Example 20: Generation and Assay of AAV Vectors Delivering CasX Constructs Targeting SOD1

This example describes a typical protocol followed to produce and characterize AAV2 vectors packaging CasX molecules and guides.

Materials and Methods:

For AAV production, the tri-plasmid transfection method was used, using three essential plasmids—pTransgene carrying the gene of interest to be packaged in AAV, pRC, and pHelper. DNA encoding CasX and guide RNA were cloned into an AAV transgene cassette, between the ITRs (FIG. 42) to generate the pTransgene plasmid. The constructed transgene plasmid was verified via full-length plasmid sequencing (see Table 15), restriction digestion, and functional tests including in vitro transfection of mammalian cells. Additional plasmids required for AAV production (pRC plasmid and pHelper plasmid) were purchased from commercial suppliers (Aldevron, Takara).

For AAV production, HEK2931T cells were cultured in FB medium in a 37° C. incubator with 5% CO2. 10-20 15 cm dishes of HEK293T cells were used in a single batch of viral production. For a single 15 cm dish, 15 ug of each plasmid was first mixed together in 4 ml of FB medium, and complexed with 145 ug polyethyleneimine (PEI) i.e., at 3 ug PEI/ug of DNA, for 10 mins at mom temperature. The ratio of the three plasmids used may be varied to further optimize virus production as needed.

The PEI-DNA complex was then slowly dripped onto the 15 cm plate of HEK293T cells, and the plate of transfected cells moved back into the incubator. The next day, the medium was changed to FB with 2% FBS (instead of 10% FBS). At 3 days post-transfection, the media from the cells may be collected to increase virus yields. At 5-6 days post-transfection medium and cells were collected. The timing of harvest may be further varied to optimize virus yield.

The cells were pelleted by centrifugation, and the medium collected from the top. Cells were lysed in a buffer with high salt content and high-salt-active nuclease for 1 h at 37° C. The cells may also be lysed using additional methods, such as sequential freeze-thaw, or chemical lysis by detergent.

The medium collected at harvest, and any medium collected at earlier time points, were treated with a 1:5 dilution of a solution containing 40% PEG8000 and 2.5M NaCl, and incubated on ice for 2h, in order to precipitate AAV. The incubation may also be carried out overnight at 4° C.

The AAV precipitate from the medium was pelleted by centrifugation, resuspended in high salt content buffer with high-salt-active nuclease and combined with the lysed cell pellet. The combined cell lysate was then clarified by centrifugation and filtration through a 0.45 um filter, and purified on an AAV Poros affinity resin column (Thermofisher Scientific). The virus was eluted from the column into a neutralizing solution. At this stage, the virus may be taken through additional rounds of purification to increase the quality of the virus preparation.

The eluted virus was then titered via qPCR to quantify the virus yield. For titering, a sample of virus was first digested with DNAse to remove any non-packaged viral DNA, the DNAse deactivated, and then viral capsids disrupted with Proteinase K to expose the packaged viral genomes for titering.

Results:

Representative titers for AAV packaging DNA encoding a CasX 119 molecule and rRNA guide 64 (119.64) with a spacer having the sequence ATGTTCATGAGTTTGGAGAT; SEQ ID NO: 239 is shown in FIG. 43. Typically, ˜1e13 viral genomes were obtained from one batch of virus production as described here.

This example demonstrates that i) CasX and a gNA can be cloned into an AAV transgene construct, and ii) CasX and guide can be packaged in an AAV vector and produced at sufficiently high titers.

TABLE 15 Sequence of pStx17 Construct Construct DNA sequence pStX17 SEQ ID NO: 3569

Example 21: Administration of AAV Vectors Encoding a CasX System In Vitro and Evidence of SOD1 Gene Editing Materials and Methods:

SOD1-GFP reporter cells were seeded at 30k cells/well in a 96 well plate in 100 μl of FB medium. Confluence of cells was checked the next day, and cells were transduced at 80% confluence with AAV vectors (packaging construct 119.64 targeting SOD1, and SauCas9 targeting SOD1) at a range of doses or multiplicity of infection (MOI), for example from 1e7 to 1 viral genomes per cell. In a separate experiment, neural progenitor cells from the G93A mouse model of ALS (G93A NPCs) were similarly transduced. NPCs are cultured in NPC medium (DMEMF12 with Glutamax, supplemented with 10 mM Hepes (100× Thermofisher #15630080), non-essential amino acids (100× Thermofisher #11140050), penicillin-streptomycin (100×-Pen-Strep; GIBCO #15140-122), 2-mercaptoethanol 1000× (Thermofisher #21985023), B27 without vitamin-A (50×, Thermofisher), N2 (100×, Thermofisher), 20 ng/ml bFGF (Biolegend Cat no #579606), and 20 ng/ml EGF (Thermofisher #PHG0311)) at 37° C. and 5% CO2. The AAV doses were calculated based on viral titers determined by qPCR. Fresh FB medium or NPC medium may be replenished the next day, or as needed. Starting at 5 days post-transduction, and weekly thereafter, a portion of the cells were analyzed via flow cytometry or T7E1 assay.

Results:

A representative example of SOD1 editing, as demonstrated by percentage of GFP negative cells, at 12 days post-transduction is shown in FIG. 44 and FIG. 45. FIG. 46 shows CasX delivered via AAV, with evidence of editing of G93A NPCs.

This example demonstrates that CasX constructs targeting SOD1 may be delivered to mammalian cells via AAV, and result in successful editing of the SOD1 locus.

Example 22: In Vitro Transcription for the Generation of Guides and Spacers

For the generation of RNA single guides and spacers, templates for in vitro transcription were generated by performing PCR with Q5 polymerase (NEB M0491) according to the recommended protocol, with template oligos for each backbone and amplification primers with the T7 promoter and the spacer sequence. The DNA primer sequences for the T7 promoter, guide and spacer for guides and spacers are presented in Table 16, below. The template oligos, labeled “backbone fwd” and “backbone rev” for each scaffold, were included at a final concentration of 20 nM each, and the amplification primers (T7 promoter and the unique spacer primer) were included at a final concentration of 1 uM each. The sg2, sg32, sg64, and sg174 guides correspond to SEQ ID NOS: 5, 2104, 2106, and 2238, respectively, with the exception that sg2, sg32, and sg64 were modified with additional 5′ G to increase transcription efficiency (compare sequences in Table 16 to Table 2). The 7.37 spacer targets beta2-microglobulin (B2M). Following PCR amplification, templates were cleaned and isolated by phenol-chloroform-isoamyl alcohol extraction followed by ethanol precipitation.

In vitro transcriptions were carried out in buffer containing 50 mM Tris pH 8.0, 30 mM MgCl₂, 0.01% Triton X-100, 2 mM spermidine, 20 mM DTT, 5 mM NTPs, 0.5 μM template, and 100 μg/mL T7 RNA polymerase. Reactions were incubated at 37° C. overnight. 20 units of DNase I (Promega #M6101)) were added per 1 mL of transcription volume and incubated for one hour. RNA products were purified via denaturing PAGE, ethanol precipitated, and resuspended in 1× phosphate buffered saline. To fold the sgRNAs, samples were heated to 70° C. for 5 min and then cooled to room temperature. The reactions were supplemented to 1 mM final MgCl₂ concentration, heated to 50° C. for 5 min and then cooled to room temperature. Final RNA guide products were stored at −80° C.

TABLE 16 Sequences Primer Sequence Primer (SEQ ID NO) SEQ ID NO RNA product T7 promoter primer 3550 NA Used for all sg2 backbone fwd 3551 3563 GGUACUGGCGCUUUUAUCUCAUUACUUUGAG sg2 backbone rev 3552 AGCCAUCACCAGCGACUAUGUCGUAUGGGUA sg2.7.37 spacer 3553 AAGCGCUUAUUUAUCGGAGAGAAAUCCGAUA primer AAUAAGAAGCAUCAAAGGGCCGAGAUGUCUC GCUCCG sg32 backbone fwd 3554 3564 GGUACUGGCCCUUUUAUCUCAUUACUUUGAG sg32 backbone rev 3555 AGCCAUCACCAGCGACUAUGUCGUAUGGGUA sg32.7.37 spacer 3556 AAGCGCCCUCUUCGGAGGGAAGCAUCAAAGG primer GCCGAGAUGUCUCG sg64 backbone fwd 3557 3565 GGUACUGGCGCCUUUAUCUCAUUACUUUGAG sg64 backbone rev 3558 AGCCAUCACCAGCGACUAUGUCGUAUGGGUA 5g64.7.37 spacer 3559 AAGCGCUUACGGACUUCGGUCCGUAAGAAGC primer AUCAAAGGGCCGAGAUGUCUCGCUCCG sg174 backbone fwd 3560 3566 ACUGGCGCUUUUAUCUgAUUACUUUGAGAGC sg174 backbone rev 3561 CAUCACCAGCGACUAUGUCGUAgUGGGUAAA sg174.7.37 spacer 3562 GCUCCCUCUUCGGAGGGAGCAUCAAAGGGCC primer GAGAUGUCUCGCUCCG

Example 23: Editing of Gene Targets PCSK9, PMP22, TRAC, SOD1, B2M and HTT

The purpose of this study was to evaluate the ability of the CasX variant 119 and gNA variant 174 to edit nucleic acid sequences in six gene targets.

Materials and Methods

Spacers for all targets except B2M and SOD1 were designed in an unbiased manner based on PAM requirements (TTC or CTC) to target a desired locus of interest. Spacers targeting B2M and SOD1 had been previously identified within targeted exons via lentiviral spacer screens carried out for these genes. Designed spacers for the other targets were ordered from Integrated DNA Technologies (IDT) as single-stranded DNA (ssDNA) oligo pairs. ssDNA spacer pairs were annealed together and cloned via Golden Gate cloning into a base mammalian-expression plasmid construct that contains the following components: codon optimized Cas X 119 protein+NLS under an EF1A promoter, guide scaffold 174 under a U6 promoter, carbenicillin and puromycin resistance genes. Assembled products were transformed into chemically-competent E. coli, plated on Lb-Agar plates (LB: Teknova Cat #L9315, Agar: Quartzy Cat #214510) containing carbenicillin and incubated at 37° C. Individual colonies were picked and miniprepped using Qiagen Qiaprep spin Miniprep Kit (Qiagen Cat #27104) following the manufacturer's protocol. The resulting plasmids were sequenced through the guide scaffold region via Sanger sequencing (Quintara Biosciences) to ensure correct ligation.

HEK 293T cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Corning Cellgro, #10-013-CV) supplemented with 10% fetal bovine serum (FBS; Seradigm, #1500-500), 100 Units/ml penicillin and 100 mg/ml streptomycin (100×-Pen-Strep; GIBCO #15140-122), sodium pyruvate (10×, Thermofisher #11360070), non-essential amino acids (100× Thermofisher #11140050), HEPES buffer (100× Thermofisher #15630080), and 2-mercaptoethanol (1000× Thermofisher #21985023). Cells were passed every 3-5 days using TryplE and maintained in an incubator at 37° C. and 5% CO2.

On day 0, HEK293T cells were seeded in 96-well, flat-bottom plates at 30k cells/well. On day 1, cells were transfected with 100 ng plasmid DNA using Lipofectamine 3000 according to the manufacturer's protocol. On day 2, cells were switched to FB medium containing puromycin. On day 3, this media was replaced with fresh FB medium containing puromycin. The protocol after this point diverged depending on the gene of interest. Day 4 for PCSK9, PMP22, and TRAC: cells were verified to have completed selection and switched to FB medium without puromycin. Day 4 for B2M, SOD1, and HTT: cells were verified to have completed selection and passed 1:3 using TryplE into new plates containing FB medium without puromycin. Day 7 for PCSK9, PMP22, and TRAC: cells were lifted from the plate, washed in dPBS, counted, and resuspended in Quick Extract (Lucigen, QE09050) at 10,000 cells/μl. Genomic DNA was extracted according to the manufacturer's protocol and stored at −20° C. Day 7 for B2M, SOD1, and HTT: cells were lifted from the plate, washed in dPBS, and genomic DNA was extracted with the Quick-DNA Miniprep Plus Kit (Zymo, D4068) according to the manufacturer's protocol and stored at −20° C.

NGS Analysis: Editing in cells from each experimental sample was assayed using next generation sequencing (NGS) analysis. All PCRs were carried out using the KAPA HiFi HotStart ReadyMix PCR Kit (KR0370). The template for genomic DNA sample PCR was 5 μl of genomic DNA in QE at 10k cells/μL for PCSK9, PMP22, and TRAC. The template for genomic DNA sample PCR was 400 ng of genomic DNA in water for B2M, SOD1, and HTT. Primers were designed specific to the target genomic location of interest to form a target amplicon. These primers contain additional sequence at the 5′ ends to introduce Illumina read and 2 sequences. Further, they contain a 7 nt randomer sequence that functions as a unique molecular identifier (UMI). Quality and quantification of the amplicon was assessed using a Fragment Analyzer DNA analyzer kit (Agilent, dsDNA 35-1500 bp). Amplicons were sequenced on the Illumina Miseq according to the manufacturer's instructions. Resultant sequencing reads were aligned to a reference sequence and analyzed for indels. Samples with editing that did not align to the estimated cut location or with unexpected alleles in the spacer region were discarded.

Results

In order to validate the editing effected by the CasX:gNA 119.174 at a variety of genetic loci, a clonal plasmid transfection experiment was performed in HEK 293T cells. Multiple spacers (Table 17) were designed and cloned into an expression plasmid encoding the CasX 119 nuclease and guide 174 scaffold. HEK 293T cells were transfected with plasmid DNA, selected with puromycin, and harvested for genomic DNA six days post-transfection. Genomic DNA was analyzed via next generation sequencing (NGS) and aligned to a reference DNA sequence for analysis of insertions or deletions (indels). CasX:gNA 119.174 was able to efficiently generate indels across the 6 target genes, as shown in FIGS. 47 and 48. Indel rates varied between spacers, but median editing rates were consistently at 60% or higher, and in some cases, indel rates as high as 91% were observed. Additionally, spacers with non-canonical CTC PAMs were demonstrated to be able to generate indels with all tested target genes (FIG. 49).

The results demonstrate that the CasX variant 119 and gNA variant 174 can consistently and efficiently generate indels at a wide variety of genetic loci in human cells. The unbiased selection of many of the spacers used in the assays shows the overall effectiveness of the 119.174 RNP molecules to edit genetic loci, while the ability to target to spacers with both a TTC and a CTC PAM demonstrates its increased versatility compared to reference CasX that edit only with the TTC PAM.

TABLE 17 Spacer sequences targeting each genetic locus. Gene Spacer PAM Spacer Sequence SEQ ID NO: PCSK9 6.1 TTC GAGGAGGACGGCCTGGCCGA 3570 PCSK9 6.2 TIC ACCGCTGCGCCAAGGTGCGG 3571 PCSK9 6.4 TTC GCCAGGCCGTCCTCCTCGGA 3572 PCSK9 6.5 TTC GTGCTCGGGTGCTTCGGCCA 3573 PCSK9 6.3 TTC ATGGCCTTCTTCCTGGCTTC 3574 PCSK9 6.6 TTC GCACCACCACGTAGGTGCCA 3575 PCSK9 6.7 TTC TCCTGGCTTCCTGGTGAAGA 3576 PCSK9 6.8 TTC TGGCTTCCTGGTGAAGATGA 3577 PCSK9 6.9 TTC CCAGGAAGCCAGGAAGAAGG 3578 PCSK9 6.10 TTC TCCTTGCATGGGGCCAGGAT 3579 PMP22 18.16 TIC GGCGGCAAGTTCTGCTCAGC 3580 PMP22 18.17 TTC TCTCCACGATCGTCAGCGTG 3581 PMP22 18.18 CTC ACGATCGTCAGCGTGAGTGC 3582 PMP22 18.1 TTC CTCTAGCAATGGATCGTGGG 3583 TRAC 15.3 TIC CAAACAAATGTGTCACAAAG 3584 TRAC 15.4 TTC GATGTGTATATCACAGACAA 3585 TRAC 15.5 TTC GGAATAATGCTGTTGTTGAA 3586 TRAC 15.9 TTC AAATCCAGTGACAAGTCTGT 3587 TRAC 15.10 TTC AGGCCACAGCACTGTTGCTC 3588 TRAC 15,21 TTC AGAAGACACCTTCTTCCCCA 3589 TRAC 15.22 TTC TCCCCAGCCCAGGTAAGGGC 3590 TRAC 15.23 TTC CCAGCCCAGGTAAGGGCAGC 3591 HTT 5.1 TTC AGTCCCTCAAGTCCTTCCAG 3592 HTT 5,2 TTC AGCAGCAGCAGCAGCAGCAG 3593 HTT 5.3 TTC TCAGCCGCCGCCGCAGGCAC 3594 HTT 5.4 TTC AGGGTCGCCATGGCGGTCTC 3595 HTT 5.5 TTC TCAGCTTTTCCAGGGTCGCC 3596 HTT 5.7 CTC GCCGCAGCCGCCCCCGCCGC 3597 HTT 5.8 CTC GCCACAGCCGGGCCGGGTGG 3598 HTT 5.9 CTC TCAGCCACAGCCGGGCCGGG 3599 HTT 5.10 CTC CGGTCGGTGCAGCGGCTCCT 3600 SOD1 8.56 TTC CCACACCTTCACTGGTCCAT 3601 SOD1 8.57 TTC TAAAGGAAAGTAATGCACCA 3602 SOD1 8.58 TTC CTGGTCCATTACTTTCCTTT 3603 SOD1 8.2 TTC ATGTTCATGAGTTTGGAGAT 239 SOD1 8.68 TTC TGAGTTTGGAGATAATACAG 3604 SOD1 8.59 TTC ATAGACACATCGGCCACACC 3605 SOD1 8.47 TTC TTATTAGGCATGTTGGAGAC 3606 SOD1 8.62 CTC CAGGAGACCATTGCATCATT 3607 B2M 7.120 TTC GGCCTGGAGGfTATCCAGCG 3608 B2M 7.37 TTC GGCCGAGATGTCTCGCTCCG 3609 B2M 7.43 CTC AGGCCAGAAAGAGAGAGTAG 3610 B2M 7.119 CTC CGCTGGATAGCCTCCAGGCC 3611 B2M 7.14 TTC TGAAGCTGACAGCATTCGGG 3612

Example 24: Design and Evaluation of Improved CasX Variants by Deep Mutational Evolution

The purpose of the experiments was to identify and engineer novel CasX protein variants with enhanced genome editing efficiency relative to wild-type CasX. To cleave DNA efficiently in living cells, the CasX protein must efficiently perform the following functions: i) form and stabilize the R-loop structure consisting of a targeting guide RNA annealed to a complementary genomic target site in a DNA:RNA hybrid; and ii) position an active nuclease domain to cleave both strands of the DNA at the target sequence. These two functions can each be enhanced by altering the biochemical or structural properties of the protein, specifically by introducing amino acid mutations or exchanging protein domains in an additive or combinatorial fashion.

To construct CasX protein variants with improved properties, an overall approach was chosen in which bacterial assays and hypothesis-driven approaches were first used to identify candidate mutations to enhance particular functions, after which increasingly stringent human genome editing assays were used in a stepwise manner to rationally combine cooperatively function-enhancing mutations in order to identify CasX variants with enhanced editing properties.

Materials and Methods: Cloning and Media

Restriction enzymes, PCR reagents, and cloning strains of E. coli were obtained from New England Biolabs. All molecular biology and cloning procedures were performed according to the manufacturer's instructions. PCR was performed using Q5 polymerase unless otherwise specified. All bacterial culture growth was performed in 2XYT media (Teknova) unless otherwise specified. Standard plasmid cloning was performed in Turbot E. coli unless otherwise specified. Standard final concentrations of the following antibiotics were used where indicated: carbenicillin: 100 μg/mL; kanamycin: 60 μg/mL; chloramphenicol: 25 μg/mL.

Molecular Biology of Protein Library Construction

Four libraries of CasX protein variants were constructed using plasmid recombineering in E. coli strain EcNR2 (Addgene ID: 26931), and the overall approach to protein mutagenesis was termed Deep Mutational Evolution (DME), which is schematically shown in FIG. 50. Three libraries were constructed corresponding to each of three cleavage-inactivating mutations made to the reference CasX protein open reading frame of Planctomycetes, SEQ ID NO:2 (“STX2”), rendering the CasX catalytically dead (dCasX). These three mutations are referred to as D1 (with a D659A substitution), D2 (with an E756A substitution), or D3 (with a D922A substitution). A fourth library was composed of all three mutations in combination, referred to as DDD (D659A; E756A; D922A substitutions). These libraries were constructed by introducing desired mutations to each of the four starting plasmids. Briefly, an oligonucleotide library was obtained from Twist Biosciences and prepared for recombineering (see below). A final volume of 50 μL of 1 μM oligonucleotides, plus 10 ng of pSTX1 encoding the dCasX open reading frame (composed of either D1, D2, or D3) was electroporated into 50 μL of induced, washed, and concentrated EcNR2 using a 1 mm electroporation cuvette (BioRad GenePulser). A Harvard Apparatus ECM 630 Electroporation System was used with settings 1800 kV, 200 Ω, 25 ρF. Three replicate electroporations were performed, then individually allowed to recover at 30° C. for 2 hr in 1 mL of SOC (Teknova) without antibiotic. These recovered cultures were titered on LB plates with kanamycin to determine the library size. 2XYT media and kanamycin was then added to a final volume of 6 mL and grown for a further 16 hours at 30° C. Cultures were miniprepped (QIAprep Spin Miniprep Kit) and the three replicates were then combined, completing a round of plasmid recombineering. A second round of recombineering was then performed, using the resulting miniprepped plasmid from round 1 as the input plasmid.

Oligo library synthesis and maturation: A total of 57751 unique oligonucleotide sequences designed to result in either amino acid insertion, substitution, or deletion at each codon position along the STX 2 open reading frame were synthesized by Twist Biosciences, among which were included so-called ‘recombineering oligos’ that included one codon to represent each of the twenty standard amino acids and codons with flanking homology when encoded in the plasmid pSTX1. The oligo library included flanking 5′ and 3′ constant regions used for PCR amplification. Compatible PCR primers include oSH7: 5′AACACGTCCGTCCTAGAACT (universal forward; SEQ ID NO: 3613) and oSH8: 5′ACTTGGTTACGCTCAACACT (universal reverse; SEQ ID NO: 3614) (see reference table). The entire oligo pool was amplified as 400 individual 100 μL reactions. The protocol was optimized to produce a clean band at 164 bp. Finally, amplified oligos were digested with a restriction enzyme (to remove primer annealing sites, which would otherwise form scars during recombineering), and then cleaned, for example, with a PCR clean-up kit (to remove excess salts that may interfere with the electroporation step). Here, a 600 μL final volume BsaI restriction digest was performed, with 30 μg DNA+30 μL BsaI enzyme, which was digested for two hours at 37° C.

For DME1: after two rounds of recombineering were completed, plasmid libraries were cloned into a bacterial expression plasmid, pSTX2. This was accomplished using a BsmbI Golden Gate Cloning approach to subclone the library of STX genes into an expression compatible context, resulting in plasmid pSTX3. Libraries were transformed into Turbo® E. coli (New England Biolabs) and grown in chloramphenicol for 16 hours at 37° C., followed by miniprep the next day.

For DME2: protein libraries from DME1 were further cloned to generate a new set of three libraries for further screening and analysis. All subcloning and PCR was accomplished within the context of plasmid pSTX1. Library D1 was discontinued and libraries D2 and D3 were kept the same. A new library, DDD, was generated from libraries D2 and D3 as follows. First, libraries D2 and D3 were PCR amplified such that the Dead 1 mutation, E756A, was added to all plasmids in each library, followed by blunt ligation, transformation, and miniprep, resulting in library A (D1+D2) and library B (D1+D3). Next, another round of PCR was performed to add either mutation D3 or D2, respectively, to library A and B, generating PCR products A′ and B′. At this point, A′ and B′ were combined in equimolar amounts, then blunt ligated, transformed, and miniprepped to generate a new library, DDD, containing all three dead mutations in each plasmid.

Bacterial CRISPR Interference (CRISPRi) Screen

A dual-color fluorescence reporter screen was implemented, using monomeric Red Fluorescent Protein (mRFP) and Superfolder Green Fluorescent Protein (sfGFP), based on Qi L S, et al. Cell 152:1173-1183 (2013). This screen was utilized to assay gene-specific transcriptional repression mediated by programmable DNA binding of the CasX system. This strain of E. coli expresses bright green and red fluorescence under standard culturing conditions or when grown as colonies on agar plates. Under a CRISPRi system, the CasX protein is expressed from an anhydrotetracycline (aTc)-inducible promoter on a plasmid containing a p15A replication origin (plasmid pSTX3; chloramphenicol resistant), and the sgRNA is expressed from a minimal constitutive promoter on a plasmid containing a ColE1 replication origin (pSTX4, non-targeting spacer, or pSTX5, GFP-targeting spacer #1; carbenicillin resistant). When the CRISPRi E. coli strain is co-transformed with both plasmids, genes targeted by the spacer in pSTX4 are repressed; in this case GFP repression is observed, the degree to which is dependent on the function of the targeting CasX protein and sgRNA. In this system, RFP fluorescence can serve as a normalizing control. Specifically, RFP fluorescence is unaltered and independent of functional CasX based CRISPRi activity. CRISPRi activity can be tuned in this system by regulating the expression of the CasX protein: here, all assays used an induction concentration of 20 nM aTc final concentration in growth media.

Libraries of CasX protein were initially screened using the above CRISPRi system. After co-transformation and recovery, libraries were either: 1) plated on LB agar plus appropriate antibiotics and titered such that individual colonies could be picked, or 2) grown for eight hours in 2XYT media with appropriate antibiotics and sorted on a MA900 flow cytometry instrument (Sony). Variants of interest were detected using either standard Sanger sequencing of picked colonies (UC Berkeley Barker Sequencing Facility) or NGS sequencing of miniprepped plasmid (Massachusetts General Hospital CCIB DNA Core Next-Generation Sequencing Service).

Plasmids were miniprepped and the protein sequence was PCR-amplified, then tagmented using a Nextera kit (Illumina) to fragment the amplicon and introduce indexing adapters for sequencing on a 150 paired end HiSeq 2500 (UC Berkeley Genomics Sequencing Lab).

Bacterial ccdB Plasmid Clearance Selection

A dual-plasmid selection system was used to assay clearance of a toxic plasmid by CasX DNA cleavage. Briefly, the arabinose-inducible plasmid pBLO63.3 expressing toxic protein ccdB results in death when transformed into E. coli strain BW25113 and grown under permissive conditions. However, growth is rescued if the plasmid is cleared successfully by dsDNA cleavage, and in particular by plasmid pSTX3 co-expressing CasX protein and a guide RNA targeting the plasmid pBLO63.3. CasX protein libraries from DME1, without the catalytically inactivating mutations D1, D2, or D3, were subcloned into plasmid pSTX3. These plasmid libraries were transformed into BW25113 carrying pBLO63.3 by electroporation (200 ng of plasmid into 50 uL of electrocompetent cells) and allowed to recover in 2 mL of SOC media at 37° C. at 200 rpm shaking for 25 minutes, after which 1 uL of 1M IPTG was added. Growth was continued for an additional 40 minutes, after which cultures were evenly divided across a 96-well deep-well block and grown in selective media for 4.5 hrs at 37° C. or 45° C. at 750 rpm. Selective media consists of the following: 2XYT with chloramphenicol+10 mM arabinose+500 μM IPTG+2 nM aTc (concentrations final). Following growth, plasmids were miniprepped to complete one round of selection, and the resulting DNA was used as input for a subsequent round. Seven rounds of selection were performed on CasX protein libraries. CasX variant Sanger sequencing or NGS was performed as described above.

NGS Data Analysis

Paired end reads were trimmed for adapter sequences with cutadapt (version 2.1), and aligned to the reference with bowtie2 (v2.3.4.3). The reference was the entire amplicon sequence prior to tagmentation in the Nextera protocol. Each catalytically inactive CasX variant was aligned to its respective amplicon sequence. Sequencing reads were assessed for amino acid variation from the reference sequence. In short, the read sequence and aligned reference sequence were translated (in frame), then realigned and amino acid variants were called. Reads with poor alignment or high error rates were discarded (mapq<20 and estimated error rate>4%; Estimated error rate was calculated using per-base phred quality scores). Mutations at locations of poor-quality sequencing were discarded (phred score<20). Mutations were labeled for being single substitutions, insertions, or deletions, or other higher-order mutations, or outside the protein-coding sequence of the amplicon. The number of reads that supported each set of mutations was determined. These read counts were normalized for sequencing depth (mean normalization), and read counts from technical replicates were averaged by taking the geometric mean. Enrichment was calculated within each CasX variant by averaging the enrichment for each gate.

Molecular Biology of Variants

In order to screen variants of interest, individual variants were constructed using standard molecular biology techniques. All mutations were built on STX2 using a staging vector and Gibson cloning. To build single mutations, universal forward (5′-+3′) and reverse (3′-+5′) primers were designed on either end of the protein sequence that had homology to the desired backbone for screening (see Table 18). Primers to create the desired mutations were also designed (F primer and its reverse complement) and used with the universal F and R primers for amplification, thus producing two fragments. In order to add multiple mutations, additional primers with overlap were designed and more PCR fragments were produced. For example, to construct a triple mutant, four sets of F/R primers were designed. The resulting PCR fragments were gel extracted and the screening vector was digested with the appropriate restriction enzymes then gel extracted. The insert fragments and vector were then assembled using Gibson assembly master mix, transformed, and plated using appropriate LB agar+antibiotic. The clones were Sanger sequenced and correct clones were chosen.

Finally, spacer cloning was performed to target the guide RNA to a gene of interest in the appropriate assay or screen. The sequence verified non-targeting clone was digested with the appropriate golden gate enzyme and cleaned using DNA Clean and Concentrator kit (Zymo). The oligos for the spacer of interest were annealed. The annealed spacer was ligated into digested and cleaned vector using a standard Golden Gate Cloning protocol. The reaction was transformed and plated on LB agar+antibiotic. The clones were sanger sequenced and correct clones were chosen.

TABLE 18 Primer sequences Screening vector F primer sequence R primer sequence pSTX6 SAH24: SAH25: TTCAGGTTGGACCGGTGCCACCATGGCCCC TTTTGGACTAGTCACGGCGGGC AAAGAAGAAGCGGAAGGTCAGCCAAGAG TTCCAG (SEQ ID NO: 3616) ATCAAGAGAATCAACAAGATCAGA (SEQ ID NO: 3615) pSTX116 or oIC539: oIC540: pSTX34 ATGGCCCCAAAGAAGAAGCGGAAGGTCTC TACCTTTCTCTTCTTTTTTGGAC TAGACAAG (SEQ ID NO: 3617) TAGTCACGG (SEQ ID NO: 3618)

GFP Editing by Plasmid Lipofection of HEK293T Cells

Either doxycycline inducible GFP (iGFP) reporter HEK293T cells or SOD1-GFP reporter HEK293T cells were seeded at 20-40k cells/well in a 96 well plate in 100 μl of FB medium and cultured in a 37° C. incubator with 5% C02. The following day, confluence of seeded cells was checked. Cells were ˜75% confluent at time of transfection. Each CasX construct was transfected at 100-500 ng per well using Lipofectamine 3000 following the manufacturer's protocol, into 3 wells per construct as replicates. SaCas9 and SpyCas9 targeting the appropriate gene were used as benchmarking controls. For each Cas protein type, a non-targeting plasmid was used as a negative control. After 24-48 hours of puromycin selection at 0.3-3 μg/ml to select for successfully transfected cells, followed by 1-7 days of recovery in FB medium, GFP fluorescence in transfected cells was analyzed via flow cytometry. In this process, cells were gated for the appropriate forward and side scatter, selected for single cells and then gated for reporter expression (Attune Nxt Flow Cytometer, Thermo Fisher Scientific) to quantify the expression levels of fluorophores. At least 10,000 events were collected for each sample. The data were then used to calculate the percentage of edited cells.

GFP Editing by Lentivirus Transduction of HEK293T Cells

Lentivirus products of plasmids encoding CasX proteins, including controls. CasX variants, and/or CasX libraries, were generated in a Lenti-X 293T Cell Line (Takara) following standard molecular biology and tissue culture techniques. Either iGFP HEK293T cells or SOD1-GFP reporter HEK293T cells were transduced using lentivirus based on standard tissue culture techniques. Selection and fluorescence analysis was performed as described above, except the recovery time post-selection was 5-21 days. For Fluorescence-Activated Cell Sorting (FACS), cells were gated as described above on a MA900 instrument (Sony). Genomic DNA was extracted by QuickExtract™ DNA Extraction Solution (Lucigen) or Genomic DNA Clean & Concentrator (Zymo).

Engineering of CasX Protein 2 to CasX 119

Prior work had demonstrated that CasX RNP complexes composed of functional wild-type CasX protein from Planctomycetes (hereafter referred to as CasX protein 2 (or STX2, or STX protein 2, SEQ ID NO: 2) and CasX sgRNA 1{or STX sgRNA 1, SEQ ID NO: 4}) are capable of inducing dsDNA cleavage and gene editing of mammalian genomes (Liu, J J et al Nature, 566, 218-223 (2019)). However, previous observations of cleavage efficiency were relatively low (˜30% or less), even under optimal laboratory conditions. These poor rates of genome editing may be insufficient for the wild-type CasX CRISPR systems to serve as therapeutic genome-editing molecules. In order to efficiently perform genome editing, the CasX protein must effectively perform two central functions: (i) form and stabilize the R-loop, and (ii) position the nuclease domain for cleavage of both DNA strands. Under conditions in which CasX RNP can access genomic DNA, genome editing rates will be partly governed by the ability of the CasX protein to perform these functions (the other controlling component being the guide RNA). The optimization of both functions is dependent on the complex sequence-function relationship between the linear chain of amino acids encoding the CasX protein and the biochemical properties of the fully formed, cleavage competent RNP. As amino acid mutations that enhance each of these functions can be combined to cumulatively result in a highly engineered CasX protein exhibiting greatly enhanced genome editing efficiency sufficient for human therapeutics, an overall engineering approach was devised in which mutations enhancing function (i) were identified, mutations enhancing function (ii) were identified, and then rational stacking (or combination) of multiple beneficial mutations would be used to construct CasX variants capable of efficient genome editing. Function (i), stabilization of the R-loop, is by itself sufficient to interfere with gene expression in living cells even in the absence of DNA nuclease activity, a phenomenon known as CRISPR interference (CRISPRi). It was determined that a bacterial CRISPRi assay would be well-suited to identifying mutations enhancing this function. Similarly, a bacterial assay testing for double-stranded DNA (dsDNA) cleavage would be capable of identifying mutations enhancing function (ii). A toxic plasmid clearance assay was chosen to serve as a bacterial selection strategy and identify relevant amino acid changes. These sets of mutations were then validated to provide an enhancement to human genome editing activity, and served as the foundation for more extensive and rational combinatorial testing across increasingly stringent assays.

The identification of mutations enhancing core functions was performed in an engineering cycle of protein library design, molecular biology construction of libraries, and high-throughput assay of the libraries. Potential improved variants of the STX2 protein were either identified by NGS of a high-throughput biological assay, sequenced directly as clones from a population, or designed de novo for specific hypothesis testing. For high-throughput assays of functions (i) or (ii), a comprehensive and unbiased design approach to mutagenesis was used for initial diversification. Plasmid recombineering was chosen as a sufficiently comprehensive and rapid method for library construction and was performed in a promoterless staging vector pSTX1 in order to minimize library bias throughout the cloning process. A comprehensive oligonucleotide pool encoded all possible single amino acid substitutions, insertions, and deletions in the STX2 sequence was constructed by DME; the first round of library construction and screening is hereafter referred to as DME1 (FIG. 50). Two high-throughput bacterial assays were chosen to identify potential improved variants from the diverse set of mutations in DME1. As discussed above, we reasoned that a CRISPRi bacterial screen would identify mutations enhancing function (i). While CRISPRi uses a catalytically inactive form of the CasX protein, many specific characteristics together influence the total enhancement of this function, such as expression efficiency, folding rate, protein stability, or stability of the R-loop (including binding affinity to the sgRNA or DNA). DME1 libraries were constructed on the dCasX mutant templates and individually screened. Screening was performed as Fluorescence-Activated Cell Sorting (FACS) of GFP repression in a previously validated dual-color CRISPRi scheme.

Results:

For each of the DME1, DME2 and DME3 libraries, the three libraries exhibited a different baseline CRISPRi activity, thereby serving as independent, yet related, screens. For each library, gates of varying stringency were drawn around the population of interest, and sorted cell populations were deep sequenced to identify CasX mutations enhancing GFP repression (FIG. 51). A second high-throughput bacterial assay was developed to assess dsDNA cleavage in E. coli by way of selection (see methods). When this assay is performed under selective conditions, a functional STX2 RNP can exhibit ˜1000- to 10,000-fold increase in colony forming units compared to nonfunctional CasX protein (FIG. 52). Multiple rounds of liquid media selections were performed for the cleavage-competent libraries of DME1. Sequential rounds of colony picking and sequencing identified mutations to enhance function (ii). Several mutations were observed with increasing frequency with prolonged selection. One mutation of note, the deletion of proline 793, was first observed in round four at a frequency of two out of 36 sequenced colonies. After round five, the frequency increased to six out of 36 sequenced colonies. In round seven, it was observed in ten out of 48 sequenced colonies. This round-over-round enrichment suggested mutations observed in these assays could potentially enhance function (ii) of the CasX protein. Selected mutations observed across these assays can be found in table 19 as follows:

TABLE 19 Selected mutations observed in bacterial assays for function (i) or (ii) Pos. Ref. Alt.* Assay 2 Q R 45 C ccdb colony 72 T S D2 CRISPRi 80 A T 37 C ccdb colony 111 R K 45 C ccdb colony 119 G C 45 C ccdb colony 121 E D 37 C ccdb colony 153 T I 37 C ccdb colony 166 R S D2 CRISPRi 203 R K 45 C ccdb colony 270 S W 37 C ccdb colony 346 D Y 45 C ccdb colony 361 D A D1 CRISPRi 385 E A D3 CRISPRi 386 E R 45 C ccdb colony 390 K R D3 CRISPRi 399 F L 45 C ccdb colony 421 A G D2 CRISPRi 433 S N 45 C ccdb colony 489 D S D3 CRISPRi 536 F S D3 CRISPRi 546 I V D2 CRISPRi 552 E A D3 CRISPRi 591 R I 37 C ccdb colony 595 E G D3 CRISPRi 636 A D D3 CRISPRi 657 — G D1 CRISPRi 661 — L D1 CRISPRi 661 — A D1 CRISPRi 663 N S D1 CRISPRi 679 S N D2 CRISPRi 695 G H 45 C ccdb colony 696 — P 45 C ccdb colony 707 A D D3 CRISPRi 708 A K 45 C ccdb colony 712 D Q 37 C ccdb colony 732 D P D1 CRISPRi 751 A S D3 CRISPRi 774 — G Dl CRISPRi 788 A W D2 CRISPRi 789 Y T D1 CRISPRi 789 Y D D2 CRISPRi 791 C M 45 C ccdb colony 792 L E 45 C ccdb colony 793 P — 45 C ccdb colony 793 — AS 45 C ccdb colony 793 P T 45 C ccdb colony 793 P — D1 CRISPRi 793 — — D2 CRISPRi 794 — PG 45 C ccdb colony 794 — PS 45 C ccdb colony 795 — AS 37 C ccdb colony 795 — AS 45 C ccdb colony 796 — AG 37 C ccdb colony 797 — AS 45 C ccdb colony 797 Y L 45 C ccdb colony 799 S A D3 CRISPRi 867 S G 45 C ccdb colony 889 — L 37 C ccdb colony 897 L M 45 C ccdb colony 922 D K D1 CRISPRi 963 Q P D2 CRISPRi 975 K Q D2 CRISPRi *substitution, insertion, or deletion, positions are indicated relative to SEQ ID NO: 2 Pos.: Position; Ref.: Reference; Alt.: Alternative

The mutations observed in the bacterial assays above were selected for their potential to enhance CasX protein functions (i) or (ii), but desirable mutations will enhance at least one function while simultaneously remaining compatible with the other. To test this, mutations were tested for their ability to improve human cell genome editing activity overall, which requires both functions acting in concert. A HEK293T GFP editing assay was implemented in which human cells containing a stably-integrated inducible GFP (iGFP) gene were transduced with a plasmid that expresses the CasX protein and sgRNA 2 with spacers to target the RNP to the GFP gene. Mutations identified in bacterial screens, bacterial selections, as well as mutations chosen de novo from biochemical hypotheses resulting from inspection of the published Cryo-EM structure of the homologous DpbCasX protein, were tested for their relative improvement to human genome editing activity as quantified relative to the parent protein STX 2 (FIG. 53), with the greatest improvement demonstrated for construct 119, shown at the bottom of FIG. 53. Several dozen of the proposed function-enhancing mutations were found to improve human cell genome editing substantially, and selected mutations from these assays can be found in Table 20 as follows:

TABLE 20 Selected single mutations observed to enhance genome editing Fold-Improvement Position Reference Alternative* (average of two GFP spacers) 379 L R 1.4 708 A K 2.13 620 T P 1.84 385 E P 1.19 857 Y R 1.95 658 I V 1.94 399 F L 1.64 404 L K 2.23 793 P — 1.23 252 Q K 1.12** *substitution, insertion, or deletion, positions relative to SEQ ID NO: 2 **calculated as the average improvement across four variants with and without the mutation

The overall engineering approach taken here relies on the central hypothesis that individual mutations enhancing each function can be additively combined to obtain greatly enhanced CasX variants with improved editing capability, which was supported by the findings as described below; e.g., CasX variant 119 (indicated by the star in FIG. 54) exhibited a 23.9-fold improvement relative to the wild-type CasX. To test this, the single mutations were first identified if they enhanced overall editing activity. Of particular note here, a substitution of the hydrophobic leucine 379 in the helical II domain to a positively charged arginine resulted in a 1.40 fold-improvement in editing activity. This mutation might provide favorable ionic interactions with the nearby phosphate backbone of the DNA target strand (between PAM-distal bp 22 and 23), thus stabilizing R-loop formation and thereby enhancing function (i). A second hydrophobic to charged mutation, alanine 708 to lysine, increased editing activity by 2.13-fold, and might provide additional ionic interactions between the RuvC domain and the sgRNA 5′ end, thus plausibly enhancing function (i) by increasing the binding affinity of the protein for the sgRNA and thereby increasing the rate of R-loop formation. The deletion of proline 793 improved editing activity by 1.23-fold by shortening a loop between an alpha helix and a beta sheet in the RuvC domain, potentially enhancing function (ii) by favorably altering nuclease positioning for dsDNA cleavage. Overall, several dozen single mutations were found to improve editing activity, including mutations identified from each of the bacterial assays as well as mutations proposed from de novo hypothesis generation. To further identify those mutations that enhanced function in a cooperative manner, rational CasX variants composed of combinations of multiple mutations were tested (FIG. 53). An initial small combinatorial set was designed and assayed, of which CasX variant 119 emerged as the overall most improved editing molecule, with a 2.8-fold improved editing efficiency compared to the STX2 wild-type protein. Variant 119 is composed of the three single mutations L379R, A708K, and [P793], demonstrating that their individual contributions to enhancement of function are additive.

SOD1-GFP Assay Development.

To assess CasX variants with greatly improved genome-editing activity, we sought to develop a more stringent genome editing assay. The iGFP assay provides a relatively facile editing target such that STX protein 2 in the assays above exhibited an average editing efficiency of 41% and 16% with GFP targeting spacers 4.76 and 4.77 respectively. As protein variants approach 2-fold or greater efficiency improvements, the assay becomes saturated. Therefore a new HEK293T cell line was developed with the GFP sequence integrated in-frame at the C-terminus of the endogenous human gene SOD1, termed the SOD1-GFP line. This cell line served as a new, more stringent, assay to measure the editing efficiency of several hundred additional CasX protein variants (FIG. 54). Additional mutations were identified from bacterial assays, including a second iteration of DME library construction and screening, as well as utilizing hypothesis-driven approaches. Further exploration of combinatorial improved variants was also performed in the SOD1-GFP assay.

In light of the SOD1-GFP assay results, measured efficiency improvements were no longer saturated, and CasX variant 119 (indicated by the star in FIG. 54) exhibited a 23.9-fold improvement relative to the wild-type CasX (average of two spacers), with several constructs exhibiting enhanced activity relative to the CasX 119 construct. Alternatively, the dynamic range of the iGFP assay could be increased (though perhaps not completely unsaturated) by reducing the baseline activity of the WT CasX protein, namely by using sgRNA variant 1 rather than 2. Under these more stringent conditions of the iGFP assay, CasX variant 119 exhibited a 15.3-fold improvement relative to the wild-type CasX using the same spacers. Intriguingly, CasX variant 119 also exhibited substantial editing activity with spacers utilizing each of the four NTCN PAM sequences, while WT CasX only edited above 1% with spacers utilizing TTCN and ATCN PAM sequences (FIG. 55), demonstrating the ability of the CasX variant to effectively edit using an expanded spectrum of PAM sequences.

CasX Function Enhancement by Extensive Combinatorial Mutagenesis.

Potential improved variants tested in the variety of assays above provided a dataset from which to select candidate lead proteins. Over 300 proteins were assessed in individual clonal assays and of these, 197 single mutations were assessed; the remaining ˜100 proteins contained combinatorial combinations of these mutations. Protein variants were assessed via three different assays (plasmid p6 by iGFP, plasmid p6 by SOD1-GFP, or plasmid p16 by SOD1-GFP). While single mutants led to significant improvements in the iGFP assay (with fraction GFP− greater than 50%), these single-mutants all performed poorly in the SOD1-GFP p6 backbone assay (fraction GFP− less than 10%). However, proteins containing multiple, stacked mutations were able to successfully inactivate GFP in this more stringent assay, indicating that stacking of improved mutations could substantially improve cleavage activity.

Individual mutations observed to enhance function often varied in their capacity to additively improve editing activity when combined with additional mutations. To rationally quantify these epistatic effects and further improve genome editing activity, a subset of mutations was identified that had each been added to a protein variant containing at least one other mutation, and where both proteins (with and without the mutation) were tested in the same experimental context (assay and spacer; 46 mutations total). To determine the effect due to that mutation, the fraction of GFP− cells was compared with and without the mutation. For each protein/experimental context, the mutation effect was quantified as: 1) substantially improving the activity (f>1.1 f₀ where f₀ is the fraction GFP− without the mutation, and f_(v) is the fraction GFP− with the mutation), 2) substantially worsening the activity (f_(v)<0.9f₀), or 3) not affecting activity (neither of the other conditions are met). An overall score per mutation was calculated (s), based on the fraction of protein/experiment contexts in which the mutation substantially improved activity, minus the fraction of contexts in which the mutation substantially worsened activity. Out of the 46 mutations obtained, only 13 were associated with consistently increased activity (s≥0.5), and 18 mutations substantially decreased activity (s≤−0.5). Importantly, the distinction between these mutations was only clear when examining epistatic interactions across a variety of variant contexts: all of these mutations had comparable activity in the iGFP assay when measured alone.

The above quantitative analysis allowed the systematic design of an additional set of highly engineered CasX proteins composed of single mutations enhancing function both individually and in combination. First, seven out of the top 13 mutations were chosen to be stacked (the other 6 variants comprised the three variants A708K, [P793] and L379R that were included in all proteins, and another two that affected redundant positions; see FIG. 14). These mutations were iteratively stacked onto three different versions of the CasX protein: CasX 119, 311, and 365; proceeding to add only one mutation (e.g., Y857R), to adding several mutations in combination. In order to maximize the combination of enhancements for both function (i) and function (ii), individual mutations were rationally chosen to maintain a diversity of biochemical properties—i.e., multiple mutations that substitute a hydrophobic residue with a negatively charged residue were avoided. The resulting ˜30 protein variants had between five and 10 individual mutations relative to STX2 (mode=7 mutations). The proteins were tested in a lipofection assay in a new backbone context (p34) with guide scaffold 64, and most showed improvement relative to protein 19. The most improved variant of this set, protein 438, was measured to be >20% improved relative to protein 119 (see Table 21 below).

Lentiviral Transduction iGFP Assay Development

As discussed above regarding the iGFP assay, enhancements to the CasX system had likely resulted in the lipofection assay becoming saturated—that is, limited by the dynamic range of the measurement. To increase the dynamic range, a new assay was designed in which many fewer copies of the CasX gene are delivered to human cells, consisting of lentiviral transductions in a new backbone context, plasmid pSTX34 (see FIG. 35). Under this more stringent delivery modality, the dynamic range was sufficient to observe the improvements of CasX protein variant 119 in the context of a further improved sgRNA, namely sgRNA variant 174. Improved variants of both the protein and sgRNA were found to additively combine to produce yet further improved CasX CRISPR systems. Protein variant 119 and sgRNA variant 174 were each measured to improve iGFP editing activity by approximately an order of magnitude when compared with wild-type CasX protein 2 (SEQ ID NO: 2) in complex with sgRNA 1 (SEQ ID NO: 4) under the lipofection iGFP assay (FIG. 56). Moreover, improvements to editing activity from the protein and sgRNA appear to stack nearly linearly; while individually substituting CasX 2 for CasX 119, or substituting sgRNA 174 for sgRNA 1, produces a ten-fold improvement, substituting both simultaneously produces at least another ten-fold improvement (FIG. 57). Notably, this range of activity improvements exceeds the dynamic range of either assay. However, the overall activity improvement can be estimated by calculating the fold change relative to the sample 2.174, which was measured precisely in both assays. The enhancement of the highly engineered CasX CRISPR system 119.174 over wild type CasX CRISPR system 2.1 resulted in a 259-fold improvement in genome editing efficiency in human cells (+/−58, propagated standard deviation, as shown in FIG. 57), supporting that, under the conditions of the assay, the engineering of both the CasX and the guide led to dramatic improvements in editing efficiency compared to wild-type CasX and guide.

Engineering of Domain Exchange Variants

One problematic limitation of mutagenesis-based directed evolution is the combinatorial increase of the numbers of possible sequences that result as one takes larger steps in sequence-space. To overcome this, the swapping of protein domains from homologous sequences of different CasX proteins was evaluated as an alternative approach. To take advantage of the phylogenetic data available for the CasX CRISPR system, alignments were made between the CasX 1 (SEQ ID NO: 1) and CasX 2 (SEQ ID NO: 2) protein sequences, and domains were annotated for exchange in the context of improved CasX protein variant 119. To benchmark CasX 119 against the top designed combinatorial CasX protein variants and the top domain exchanged variants, all within the context of improved sgRNA 174, a stringent iGFP lentiviral transduction assay was performed. Protein variants from each class were identified as improved relative to CasX variant 119 (FIG. 58), and fold changes are represented in Table 21. For example, at day 13, CasX 119.174 with GFP spacer 4.76 leads to phenotype disruption in only ˜60% of cells, while CasX variant 491 in the same context results in >90% phenotypic editing. To summarize, the compared proteins contained the following number of mutations relative to the WT CasX protein 2: 119=3 point mutations; 438=7 point mutations; 488=protein 119, with NTSB and helical Ib domains from CasX 1 (67 mutations total); 491=5 point mutations, with NTSB and helical Ib domains from CasX 1 (69 mutations total).

TABLE 21 CasX variant improvements over CasX variant 119 in the iGFP lentiviral transduction assay, in the context of improved sgRNA 174. Fold-change Fold-change Cas X editing activity, editing activity, Protein spacer 4.76* spacer 4.77* 119 1.00 1.00 438 1.22 1.21 488 1.41 2.43 491 1.55 3.03 *relative to CasX 119

The results demonstrate that the application of rationally-designed libraries, screening, and analysis methods into a technique we have termed Deep Mutational Evolution to scan fitness landscapes of both the CasX protein and guide RNA enabled the identification and validation of mutations which enhanced specific functions, contributing to the improvement of overall genome editing activity. These datasets enabled the rational combinatorial design of further improved CasX and guide variants disclosed herein.

Example 25: Design and Evaluation of Improved Guide RNA Variants

The existing CasX platform based on wild-type sequences for dsDNA editing in human cells achieves very low efficiency editing outcomes when compared with alternative CRISPR systems (Liu, J J et al Nature, 566, 218-223 (2019)). Cleavage efficiency of genomic DNA is governed, in large part, by the biochemical characteristics of the CasX system, which in turn arise from the sequence-function relationship of each of the two components of a cleavage-competent CasX RNP: a CasX protein complexed with a sgRNA. The purpose of the following experiments was to create and identify gRNA scaffold variants with enhanced editing properties relative to wild-type CasX:gNA RNP through a program of comprehensive mutagenesis and rational approaches.

Methods

Methods for High-Throughput sgRNA Library Screens 1) Molecular Biology of sgRNA Library Construction

To build a library of sgRNA variants, primers were designed to systematically mutate each position encoding the reference gRNA scaffold of SEQ ID NO: 5, where mutations could be substitutions, insertions, or deletions. In the following in vivo bacterial screens for sgRNA mutations, the sgRNA (or mutants thereof) was expressed from a minimal constitutive promoter on the plasmid pSTX4. This minimal plasmid contains a ColE1 replication origin and carbenicillin antibiotic resistance cassette, and is 2311 base pairs in length, allowing standard Around-the-Horn PCR and blunt ligation cloning (using conventional methodologies). Forward primers KST223-331 and reverse primers KST332-440 tile across the sgRNA sequence in one base-pair increments and were used to amplify the vector in two sequential PCR steps. In step 1, 108 parallel PCR reactions were performed for each type of mutation, resulting in single base mutations at each designed position. Three types of mutations were generated. To generate base substitution mutations, forward and reverse primers were chosen in matching pairs beginning with KST224+KST332. To generate base insertion mutations, forward and reverse primers were chosen in matching pairs beginning with KST223+KST332. To generate base deletion mutations, forward and reverse primers were chosen in matching pairs beginning with KST225+KST332. After Step 1 PCR, samples were pooled into an equimolar manner, blunt-ligated, and transformed into Turbo E. coli (New England Biolabs), followed by plasmid extraction the next day. The resulting plasmid library theoretically contained all possible single mutations. In Step 2, this process of PCR and cloning was then repeated using the Step 1 plasmid library as the template for the second set of PCRs, arranged as above, to generate all double mutations. The single mutation library from Step 1 and the double mutation library from Step 2 were pooled together.

After the above cloning steps, the library diversity was assessed with next generation sequencing (see below section for methods) (see FIG. 59). It was confirmed that the majority of the library contained more than one mutation (‘other’) category. A substantial fraction of the library contained single base substitutions, deletions, and insertions (average representation within the library of 1/18,000 variants for single substitutions, and up to 1/740 variants for single deletions).

2) Assessing Library Diversity with Next Generation Sequencing.

For NGS analysis, genomic DNA was amplified via PCR with primers specific to the scaffold region of the bacterial expression vector to form a target amplicon. These primers contain additional sequence at the 5′ ends to introduce Illumina read (see Table 22 for sequences). Typical PCR conditions were: 1× Kapa Hifi buffer, 300 nM dNTPs, 300 nM each primer, 0.75 ul of Kapa Hifi Hotstart DNA polymerase in a 50 μl reaction. On a thermal cycler, incubate for 95° C. for 5 min; then 16-25 cycles of 98° C. for 15 s, 60° C. for 20 s, 72° C. for 1 min; with a final extension of 2 min at 72° C. Amplified DNA product was purified with Ampure XP DNA cleanup kit, with elution in 30 μl of water. A second PCR step was done with indexing adapters to allow multiplexing on the Illumina platform. 20 μl of the purified product from the previous step was combined with 1× Kapa GC buffer, 300 nM dNTPs, 200 nM each primer, 0.75 μl of Kapa Hifi Hotstart DNA polymerase in a 50 μl reaction. On a thermal cycler, cycle for 95° C. for 5 min; then 18 cycles of 98° C. for 15 s, 65° C. for 15 s, 72° C. for 30 s; with a final extension of 2 min at 72° C. Amplified DNA product was purified with Ampure XP DNA cleanup kit, with elution in 30 μl of water. Quality and quantification of the amplicon was assessed using a Fragment Analyzer DNA analyzer kit (Agilent, dsDNA 35-1500 bp).

TABLE 22 primer sequences. Primer SEQ ID NO PCR1 Fwd 3619 PCR2 Rvs 3620 PCR2 Fwd 3621 PCR2 Rvs v1 001 3622 PCR2_Rvs_v1_002 4294 PCR2_Rvs_v1_003 4295 PCR2_Rvs_v1_004 4296 PCR2_Rvs_v1_005 4297 PCR2_Rvs_v1_006 4298 PCR2_Rvs_v1_007 4299 PCR2_Rvs_v1_008 4300 PCR2_Rvs_v1_009 4301 PCR2_Rvs_v1_010 4302 PCR2_Rvs_v1_011 4303 PCR2_Rvs_v1_012 4304 PCR2_Rvs_v1_013 4305 PCR2_Rvs_v1_014 4306 PCR2_Rvs_v1_015 4307 PCR2_Rvs_v1_016 4308 PCR2_Rvs_v1_017 4309 PCR2_Rvs_v1_018 4310 PCR2_Rvs_v1_019 4311 PCR2_Rvs_v1_020 4312 PCR2_Rvs_v1_021 4313 PCR2_Rvs_v1_022 4314 PCR2_Rvs_v1_023 4315 PCR2_Rvs_v1_024 4316 PCR2_Rvs_v1_025 4317 PCR2_Rvs_v1_026 4318 PCR2_Rvs_v1_027 4319 PCR2_Rvs_v1_028 4320 PCR2_Rvs_v1_029 4321 PCR2_Rvs_v1_030 4322 PCR2_Rvs_v1_031 4323 PCR2_Rvs_v1_032 4324 PCR2_Rvs_v1_033 4325 PCR2_Rvs_v1_034 4326 PCR2_Rvs_v1_035 4327 PCR2_Rvs_v1_036 4328 PCR2_Rvs_v1_037 4329 PCR2_Rvs_v1_038 4330 PCR2_Rvs_v1_039 4331 PCR2_Rvs_v1_040 4332 PCR2_Rvs_v1_041 4333 PCR2_Rvs_v1_042 4334 PCR2_Rvs_v1_043 4335 PCR2_Rvs_v1_044 4336 PCR2_Rvs_v1_045 4337 PCR2_Rvs_v1_046 4338 PCR2_Rvs_v1_047 4339 PCR2_Rvs_v1_048 4340 PCR2_Rvs_v2_001 4341 PCR2_Rvs_v2_002 4342 PCR2_Rvs_v2_003 4343 PCR2_Rvs_v2_004 4344 PCR2_Rvs_v2_005 4345 PCR2_Rvs_v2_006 4346 PCR2_Rvs_v2_007 4347 PCR2_Rvs_v2_008 4348 PCR2_Rvs_v2_009 4349 PCR2_Rvs_v2_010 4350 PCR2_Rvs_v2_011 4351 PCR2_Rvs_v2_012 4352 PCR2_Rvs_v2_013 4353 PCR2_Rvs_v2_014 4354 PCR2_Rvs_v2_015 4355 PCR2_Rvs_v2_016 4356 PCR2_Rvs_v2_017 4357 PCR2_Rvs_v2_018 4358 PCR2_Rvs_v2_019 4359 PCR2_Rvs_v2_020 4360 PCR2_Rvs_v2_021 4361 PCR2_Rvs_v2_022 4362 PCR2_Rvs_v2_023 4363 PCR2_Rvs_v2_024 4364 PCR2_Rvs_v2_025 4365 PCR2_Rvs_v2_026 4366 PCR2_Rvs_v2_027 4367 PCR2_Rvs_v2_028 4368 PCR2_Rvs_v2_029 4369 PCR2_Rvs_v2_030 4370 PCR2_Rvs_v2_031 4371 PCR2_Rvs_v2_032 4372 PCR2_Rvs_v2_033 4373 PCR2_Rvs_v2_034 4374 PCR2_Rvs_v2_035 4375 PCR2_Rvs_v2_036 4376 PCR2_Rvs_v2_037 4377 PCR2_Rvs_v2_038 4378 PCR2_Rvs_v2_039 4379 PCR2_Rvs_v2_040 4380 PCR2_Rvs_v2_041 4381 PCR2_Rvs_v2_042 4382 PCR2_Rvs_v2_043 4383 PCR2_Rvs_v2_044 4384 PCR2_Rvs_v2_045 4385 PCR2_Rvs_v2_046 4386 PCR2_Rvs_v2_047 4387 PCR2_Rvs_v2_048 4388 PCR2_fwd_v1_univ 4389 PCR2_fwd_v2_univ 4390 PCR2_fwd_v2_001 4391 PCR2_fwd_v2_002 4392 PCR2_fwd_v2_003 4393 PCR2_fwd_v2_004 4394 PCR2_fwd_v2_005 4395 PCR2_fwd_v2_006 4396 PCR2_fwd_v2_007 4397 PCR2_fwd_v2_008 4398 PCR2_fwd_v2_009 4399 PCR2_fwd_v2_010 4400 PCR2_fwd_v2_011 4401 PCR2_fwd_v2_012 4402

3) Bacterial CRISPRi (CRISPR Interference) Assay

A dual-color fluorescence reporter screen was implemented, using monomeric Red Fluorescent Protein (mRFP) and Superfolder Green Fluorescent Protein (sfGFP), based on Qi L S, et al. (Cell 152, 5, 1173-1183 (2013)). This screen was utilized to assay gene-specific transcriptional repression mediated by programmable DNA binding of the CasX system). This strain of E. coli expresses bright green and red fluorescence under standard culturing conditions or when grown as colonies on agar plates. Under a CRISPRi system, the CasX protein is expressed from an anhydrotetracycline (aTc)-inducible promoter on a plasmid containing a p15A replication origin (plasmid pSTX3; chloramphenicol resistant), and the sgRNA is expressed from a minimal constitutive promoter on a plasmid containing a ColE1 replication origin (pSTX4, non-targeting spacer, or pSTX5, GFP-targeting spacer #1; carbenicillin resistant). When the E. coli strain is co-transformed with both plasmids, genes targeted by the spacer in pSTX4 are repressed; in this case GFP repression is observed, the degree to which is dependent on the function of the targeting CasX protein and sgRNA. In this system, RFP fluorescence can serve as a normalizing control. Specifically, RFP fluorescence should be unaltered and independent of functional CasX based CRISPRi activity. CRISPRi activity can be tuned in this system by regulating the expression of the CasX protein; here, all assays used an induction concentration of 20 nM anhydrotetracycline (aTc) final concentration in growth media.

Libraries of sgRNA were constructed to assess the activity of sgRNA variants in complex with three cleavage-inactivating mutations made to the reference CasX protein open reading frame of Planctomycetes, SEQ ID NO: 2, rendering the CasX catalytically dead (dCasX). These three mutations are referred to as D1 (with a D659A substitution), D2 (with a E756A substitution), or D3 (with a D922A substitution). A fourth library, composed of all three mutations in combination is referred to as DDD (D659A; E756A; D922A substitutions).

Libraries of sgRNA were screened for activity using the above CRISPRi system with either D2, D3, or DDD. After co-transformation and recovery, libraries were grown for 8 hours in 2xyt media with appropriate antibiotics and sorted on a Sony MA900 flow cytometry instrument. Each library version was sorted with three different gates (in addition to the naive, unsorted library). Three different sort gates were employed to extract GFP− cells: 10%, 1%, and “F” which represents ˜0.1% of cells, ranked by GFP repression. Finally, each sort was done in two technical replicates. Variants of interest were detected using either Sanger sequencing of picked colonies (UC Berkeley Barker Sequencing Facility) or NGS sequencing of miniprepped plasmid (Massachusetts General Hospital CCIB DNA Core Next-Generation Sequencing Service) or NGS sequencing of PCR amplicons, produced with primers that introduced indexing adapters for sequencing on an Illumina platform (see section above). Amplicons were sent for sequencing with Novogene (Beijing, China) for sequencing on an Illumina Hiseq, with 150 cycle, paired-end reads. Each sorted sample had at least 3 million reads per technical replicate, and at least 25 million reads for the naive samples. The average read count across all samples was 10 million reads.

4) NGS Data Analysis

Paired end reads were trimmed for adapter sequences with cutadapt (version 2.1), merged to form a single read with flash2 (v2.2.00), and aligned to the reference with bowtie2 (v2.3.4.3). The reference was the entire amplicon sequence, which includes ˜30 base pairs flanking the Planctomyces reference guide scaffold from the plasmid backbone having the sequence:

(SEQ ID NO: 3623) TGACAGCTAGCTCAGTCCTAGGTATAATACTAGTTACTGGCGCTTTTATC TCATTACTTTGAGAGCCATCACCAGCGACTATGTCGTATGGGTAAAGCGC TTATTTATCGGAGAGAAATCCGATAAATAAGAAGCATCAAAGCTGGAGTT GTCCCAATTCTTCTAGAG

Variants between the reference and the read were determined from the bowtie2 output. In brief, custom software in python (analyzeDME/bin/bam_to_variants.py) extracted single-base variants from the reference sequence using the cigar string and md string from each alignment. Reads with poor alignment or high error rates were discarded (mapq<20 and estimated error rate>4%; estimated error rate was calculated using per-base phred quality scores). Single-base variants at locations of poor-quality sequencing were discarded (phred score<20). Immediately adjacent single-base variants were merged into one mutation that could span multiple bases. Mutations were labeled for being single substitutions, insertions, or deletions, or other higher-order mutations, or outside the scaffold sequence.

The number of reads that supported each set of mutations was determined. These read counts were normalized for sequencing depth (mean normalization), and read counts from technical replicates were averaged by taking the geometric mean.

To obtain enrichment values for each scaffold variant, the number of normalized reads for each sorted sample were compared to the average of the normalized read counts for D2 and D3, which were highly correlated (FIG. 59B). The naive DDD sample was not sequenced. To obtain the enrichment for each catalytically dead CasX variant, the log of the enrichment values across the three sort gates were averaged.

Methods for Individual Validation of sgRNA Activity in Human Cell Assays 1) Individual sgRNA Variant Construction

In order to screen variants of interest, individual variants were constructed using standard molecular biology techniques. All mutations were built on the reference CasX (SEQ ID NO: 2) using a staging vector and Gibson cloning. To build single mutations, a universal forward (5′→3′) and reverse (3′→5′) primer were designed on either end of the encoded protein sequence that had homology to the desired backbone for screening (see Table 23 below). Primers to create the desired mutations were also designed (F primer and its reverse complement) and used with the universal F and R primers for amplification; thus producing two fragments. In order to add multiple mutations, additional primers with overlap were designed and more PCR fragments were produced. For example, to construct a triple mutant, four sets of F/R primers were designed. The resulting PCR fragments were gel extracted. These fragments were subsequently assembled into a screening vector (see Table 23), by digesting the screening vector backbone with the appropriate restriction enzymes and gel extraction. The insert fragments and vector were then assembled using Gibson assembly master mix, transformed, and plated using appropriate LB agar+antibiotic. The clones were Sanger sequenced and correct clones were chosen.

Finally, spacer cloning was performed to target the guide RNA to a gene of interest in the appropriate assay or screen. The sequence-verified non-targeting clone was digested with the appropriate Golden Gate enzyme and cleaned using DNA Clean and Concentrator kit (Zymo). The oligos for the spacer of interest were annealed. The annealed spacer was ligated into a digested and cleaned vector using a standard Golden Gate Cloning protocol. The reaction was transformed into Turbo E. coli and plated on LB agar+carbenicillin, and allowed to grow overnight at 37° C. Individual colonies were picked the next day, grown for eight hours in 2XYT+carbenicillin at 37° C., and miniprepped. The clones were Sanger sequenced and correct clones were chosen.

TABLE 23 screening vectors and associated primer sequences Screening vector F primer sequence R primer sequence pSTX6 SAH24: SAH25: TTCAGGTTGGACCGGTGCCACCATGGCCCCAAA TTTTGGACTAGTCACGGCGGGCT GAAGAAGCGGAAGGTCAGCCAAGAGATCAAGAG TCCAG (SEQ ID NO: 3616) AATCAACAAGATCAGA (SEQ ID NO: 3615) pSTX16 or oiC539: oIC540: pSTX34 ATGGCCCCAAAGAAGAAGCGGAAGGTCtctaga TACCTTTCTCTTCTTTTTTGGAC CAAG (SEG ID NO: 3617) TAGTCACGG (SEG ID NO: 3618)

2) GFP Editing by Plasmid Lipofection of HEK293T Cells

Either doxycycline-inducible GFP (iGFP) reporter HEK293T cells or SOD1-GFP reporter HEK293T cells were seeded at 20-40k cells/well in a 96 well plate in 100 μl of FB medium and cultured in a 37° C. incubator with 5% CO₂. The following day, confluence of seeded cells was checked. Cells were ˜75% confluent at time of transfection. Each CasX construct was transfected at 100-500 ng per well using Lipofectamine 3000 following the manufacturer's protocol, into 3 wells per construct as replicates. SaCas9 and SpyCas9 targeting the appropriate gene were used as benchmarking controls. For each Cas protein type, a non-targeting plasmid was used as a negative control.

After 24-48 hours of puromycin selection at 0.3-3 μg/ml to select for successfully transfected cells, followed by 1-7 days of recovery in FB medium, GFP fluorescence in transfected cells was analyzed via flow cytometry. In this process, cells were gated for the appropriate forward and side scatter, selected for single cells and then gated for reporter expression (Attune Nxt Flow Cytometer, Thermo Fisher Scientific) to quantify the expression levels of fluorophores. At least 10,000 events were collected for each sample. The data were then used to calculate the percentage of edited cells.

3) GFP Editing by Lentivirus Transduction of HEK293T Cells

Lentivirus products of plasmids encoding CasX proteins, including controls, CasX variants, and/or CasX libraries, were generated in a Lenti-X 293T Cell Line (Takara) following standard molecular biology and tissue culture techniques. Either iGFP HEK293T cells or SOD1-GFP reporter HEK293T cells were transduced using lentivirus based on standard tissue culture techniques. Selection and fluorescence analysis was performed as described above, except the recovery time post-selection was 5-21 days. For Fluorescence-Activated Cell Sorting (FACS), cells were gated as described above on a MA900 instrument (Sony). Genomic DNA was extracted by QuickExtract™ DNA Extraction Solution (Lucigen) or Genomic DNA Clean & Concentrator (Zymo).

Results:

Engineering of sgRNA 1 to 174 1) sgRNA Derived from Metagenomics of Bacterial Species Improved Function in Human Cells

An initial improvement in CasX RNP cleavage activity was found by assessing new metagenomic bacterial sequences for possible CasX guide scaffolds. Prior work demonstrated that Deltaproteobacteria sgRNA (SEQ ID NO: 4) could form a functional RNA-guided nuclease complex with CasX proteins, including the Deltaproteobactera CasX (SEQ ID NO:1 or Planctomycetes CasX (SEQ ID NO: 2). Structural characterization of this complex allowed identification of structural elements within the sgRNA (FIGS. 60A-60C). However, a sgRNA scaffold from Planctomycetes was never tested. A second tracrRNA was identified from Planctomycetes, which was made into an sgRNA with the same method as was used for Deltaproteobacteria tracrRNA-crRNA (SEQ ID NO: 5) (Liu, J J et al Nature, 566, 218-223 (2019)). These two sgRNA had similar structural elements, based on RNA secondary structure prediction algorithms, including three stem loop structures and possible triplex formation (FIG. 61).

Characterization the activity of Planctomycetes CasX protein complexed with the Deltaproteobacteria sgRNA (hereafter called RNP 2.1, wherein the CasX protein has the sequence of SEQ ID NO: 2) and Planctomycetes CasX protein complexed with scaffold 2 sgRNA (hereafter called RNP 2.2) showed clear superiority of RNP 2.2 compared to the others in a GFP-lipofection assay (see Methods) (FIG. 62). Thus, this scaffold formed the basis of our molecular engineering and optimization.

2) Improving Activity of CasX RNP Through Comprehensive RNA Scaffold Mutagenesis Screen.

To find mutations to the guide RNA scaffold that could improve dsDNA cleavage activity of the CasX RNP, a large diversity of insertions, deletions and substitutions to the gRNA scaffold 2 were generated (see Methods). This diverse library was screened using CRISPRi to determine variants that improved DNA-binding capabilities and ultimately improved cleavage activity in human cells. The library was generated through a process of pooled primer cloning as described in the Materials and Methods. The CRISPRi screen was carried out using three enzymatically-inactive versions of CasX (called D2, D3, and DDD; see Methods). Library variants with improved DNA binding characteristics were identified through a high-throughput sorting and sequencing approach. Scaffold variants from cells with high GFP repression (i.e., low fluorescence) were isolated and identified with next generation sequencing. The representation of each variant in the GFP− pool was compared to its representation in the naive library to form an enrichment score per variant (see Materials and Methods). Enrichment was reproducible across the three catalytically dead-CasX variants (FIG. 64).

Examining the enrichment scores of all single variants revealed mutable locations within the guide scaffold, especially the extended stem (FIGS. 63A-63C). The top-20 enriched single variants outside of the extended stem are listed in Table 24. In addition to the extended stem, these largely cluster into four regions: position 55 (scaffold stem bubble), positions 15-19 (triplex loop), position 27 (triplex), and in the 5′ end of the sequence (positions 1, 2, 4, 8). While the majority of these top-enriched variants were consistently enriched across all three catalytically dead CasX versions, the enrichment at position 27 was variable, with no evident enrichment in the D3 CasX (data not shown).

The enrichment of different structural classes of variants suggested that the RNP activity might be improved by distinct mechanisms. For example, specific mutations within the extended stem were enriched relative to the WT scaffold. Given that this region does not substantially contact the CasX protein (FIG. 60A), we hypothesize that mutating this region may improve the folding stability of the gRNA scaffold, while not affecting any specific protein-binding interaction interfaces. On the other hand, 5′ mutations could be associated with increased transcriptional efficiency. In a third mechanism, it was reasoned that mutations to the scaffold stem bubble or triplex could lead to increased stability through direct contacts with the CasX protein, or by affecting allosteric mechanisms with the RNP. These distinct mechanisms to improve RNP binding support that these mutations could be stacked or combined to additively improve activity.

TABLE 24 Top enriched single-variants outside of extended stem. log2 Position Annotation Reference Alternate enrichment Region 55 insertion — G 2.37466 scaffold stem bubble 55 insertion — T 1.93584 scaffold stem bubble 15 insertion — T 1.65155 triplex loop 17 insertion — T 1.56605 triplex loop 4 deletion T — 1.48676 5′ end 27 insertion — C 1.26385 triplex 16 insertion — C 1.26025 triplex loop 19 insertion — T 1.25306 triplex loop 18 insertion — G 1.22628 triplex loop 2 deletion A — 1.17690 5′ end 17 insertion — A 1.16081 triplex loop 18 substitution C T 1.10247 triplex loop 18 insertion — A 1.04716 triplex loop 16 substitution C T 0.97399 triplex loop 8 substitution G C 0.95127 pseudoknot 16 substitution C A 0.89373 triplex loop 27 insertion — A 0.86722 triplex 1 substitution T C 0.83183 5′ end 18 deletion C — 0.77641 triplex loop 19 insertion — G 0.76838 triplex loop 3) Assessing RNA Scaffold Mutants in dsDNA Cleavage Assay in Human Cells

The CRISPRi screen is capable of assessing binding capacity in bacterial cells at high throughput. However it does not guarantee higher cleavage activity in human cell assays. We next assessed a large swath of individual scaffold variants for cleavage capacity in human cells using a plasmid lipofection in HEK cells (see Materials and Methods). In this assay, human HEK293T cells containing a stably-integrated GFP gene were transduced with a plasmid (p16) that expresses reference CasX protein (Stx2) (SEQ ID NO: 2) and sgRNA comprising the gRNA scaffold variant and spacers 4.76 (having sequence UGUGGUCGGGGUAGCGGCUG (SEQ ID NO: 3624) and 4.77 (having sequence UCAAGUCCGCCAUGCCCGAA (SEQ ID NO: 3625)) to target the RNP to knockdown the GFP gene. Percent GFP knockdown was assayed using flow cytometry. Over a hundred scaffold variants were tested in this assay.

The assay resulted in largely reproducible values across different assay dates for spacer 4.76, while exhibiting more variability for spacer 4.77 (FIG. 69). Spacer 4.77 was generally less active for the wild-type RNP complex, and the lower overall signal may have contributed to this increased variability. Comparing the cleavage activity across the two spacers showed generally correlated results (r=0.652; FIG. 70). Because of the increased noise in spacer 4.77 measurements, the reported cleavage activity per scaffold was taken as the weighted average between the measurements on each scaffold, with the weights equal to the inverse squared error. This weighting effectively down-weights the contribution from high-error measurements.

A subset of sequences was tested in both the HEK-iGFP assay and the CRISPRi assay. Comparing the CRISPRi enrichment score to the GFP cleavage activity showed that highly-enriched variants had cleavage activity at or exceeding the wildtype RNP (FIG. 63C). Two variants had high cleavage activity with low enrichment scores (C18G and T17G); interestingly, these substitutions are at the same position as several highly-enriched insertions (FIG. 7I).

Examining all scaffolds tested in the HEK-iGFP assay revealed certain features that consistently improved cleavage activity. We found that the extended stem could often be completely swapped out for a different stem, with either improved or equivalent activity (e.g., compare scaffolds of SEQ ID NO: 2101-2105, 2111, 2113, 2115; all of which have replaced the extended stem, with increased activity relative to the reference, as seen in Table 5). We specifically focused on two stems with different origins: a truncated version of the wildtype stem, with the loop sequence replaced by the highly stable UUCG tetraloop (stem 42). The other (stem 46) was derived from Uvsx bacteriophage T4 mRNA, which in its biological context is important for regulation of reverse transcription of the bacteriophage genome (Tuerk et al. Proc Natl Acad Sci USA. 85(5):1364 (1988)). The top-performing gRNA scaffolds all had one of these two extended stem versions (e.g., SEQ ID NOS: 2160 and 2161).

Appending ribozymes to the 3′ end often resulted in functional scaffolds (e.g., see SEQ ID NO: 2182 with equivalent activity to the WT guide in this assay {Table 5}). On the other hand, adding to the 5′ end generally hurt cleavage activity. The best-performing 5′ ribozyme construct (SEQ ID NO: 2208) had cleavage activity<40% of the WT guide in the assay.

Certain single-point mutations were generally good, or at least not harmful, including T10C, which was designed to increase transcriptional efficiency in human cells by removing the four consecutive T's at the 5′ start of the scaffold (Kiyama and Oishi, Nucleic Acids Res., 24:4577 (1996)). C18G was another helpful mutation, which was obtained from individual colony picking from the CRISPRi screen. The insertion of C at position 27 was highly-enriched in two out of the three dCasX versions of the CRISPRi screen. However, it did not appear to help cleavage activity. Finally, insertion at position 55 within the RNA bubble substantially improved cleavage activity (i.e., compare SEQ ID NO: 2236, with a {circumflex over ( )}G55 insertion to SEQ ID NO: 2106 in Table 5).

4) Further Stacking of Variants in Higher-Stringency Cleavage Assays

Scaffold mutations that proved beneficial were stacked together to form a set of new variants that were tested under more stringent criteria: a plasmid lipofection assay in human HEK-293t cells with the GFP gene knocked into the SOD1 allele, which we observed was generally harder to knock down. Of this batch of variants, guide scaffold 158 was identified as a top-performer (FIG. 65). This scaffold had a modified extended stem (Uvsx), with additional mutations to fully base pair the extended stem ([A99] and G65U). It also contained mutations in the triplex loop (C18G) and in the scaffold stem bubble ({circumflex over ( )}G55).

In a second validation of improved DNA editing capacity, sgRNAs were delivered to cells with low-MOI lentiviral transduction, and with distinct targeting sequences to the SOD1 gene (see Methods); spacers were 8.2 (having sequence AUGUUCAUGAGUUUGGAGAU (SEQ ID NO: 3626)), and 8.4 (having sequence UCGCCAUAACUCGCUAGGCC (SEQ ID NO: 3627)) (results shown in FIG. 66). Additionally, 5′ truncations of the initial GT of guide scaffolds 158 and 64 were deleted (forming scaffolds 174 and 175 respectively). This assay showed dominance of guide scaffold 174: the variant derived from guide scaffold 158 with 2 bases truncated from the 5′ end (FIG. 66). A schematic of the secondary structure of scaffold 174 is shown in FIG. 67.

In sum, our improved guide scaffold 174 showed marked improvement over our starting reference guide scaffold (scaffold 1 from Deltaproteobacteria, SEQ ID NO:4), and substantial improvement over scaffold 2 (SEQ ID NO: 5) (FIG. 68). This scaffold contained a swapped extended stem (replacing 32 bases with 14 bases), additional mutations in the extended stem ([A99] and G65U), a mutation in the triplex loop (C18G), and in the scaffold stem bubble ({circumflex over ( )}G55) (where all the numbering refers to the scaffold 2). Finally, the initial T was deleted from scaffold 2, as well as the G that had been added to the 5′ end in order to enhance transcriptional efficiency. The substantial improvements seen with guide scaffold 174 came collectively from the indicated mutations.

Example 26: Editing of RHO in ARPE19 RHO-GFP Cells

The purpose of the experiment was to demonstrate the ability of CasX to edit the RHO locus using the CasX variants 438, 488 and 491, guide 174 variant, and spacers targeting Exon 1 of the RHO gene. Spacers were chosen based on PAM availability in the locus without prior knowledge of potential activity.

To facilitate assessment of editing outcomes, an ARPE19 RHO-GFP reporter cell line was first generated by knocking into ARPE19 cells a transgene cassette that constitutively expresses Exon 1 of the human RHO gene linked to GFP. The modified cells were expanded by serial passage every 3-5 days and maintained in Fibroblast (FB) medium, consisting of Dulbecco's Modified Eagle Medium (DMEM; Corning Cellgro, #10-013-CV) supplemented with 10% fetal bovine serum (FBS; Seradigm, #1500-500), and 100 Units/mL penicillin and 100 mg/mL streptomycin (100×-Pen-Strep; GIBCO #15140-122), and can additionally include sodium pyruvate (100×, Thermofisher #11360070), non-essential amino acids (100× Thermofisher #11140050), HEPES buffer (100× Thermofisher #15630080), and 2-mercaptoethanol (1000× Thermofisher #21985023). The cells were incubated at 37° C. and 5% CO2. After 1-2 weeks, GFP+ cells were bulk sorted into FB medium. The reporter lines were expanded by serial passage every 3-5 days and maintained in FB medium in an incubator at 37° C. and 5% CO2. Reporter clones were generated by a limiting dilution method. The clonal lines were characterized via flow cytometry, genomic sequencing, and functional modification of the RHO locus using a previously validated RHO targeting CasX molecule. The optimal reporter lines were identified as ones that i) had a single copy of GFP correctly integrated per cell, ii) maintained doubling times equivalent to unmodified cells, and iii) resulted in reduction in GFP fluorescence upon disruption of the RHO gene when assayed using the methods described below.

ARPE19 RHO-GFP reporter cells, constructed using cell line generation methods described above, were used for this experiment. Cells were seeded at 20-40k cells/well in a 96 well plate in 100 μL of FB medium and cultured in a 37° C. incubator with 5% CO2. The following day, lentiviral vectors packaging each CasX and guide construct (e.g., see Table 25 for sequences) were used to transduce cells at a high multiplicity of infection (MOI), using 3 wells per construct as replicates. A lentivirus packaging a non-targeting construct was used as a negative control. Cells were selected for successful transduction with puromycin at 0.3-3 μg/ml for 24-48 hours followed by recovery in FB medium. Edited cells were analyzed by flow cytometry 14 days after transduction. Briefly, cells were sequentially gated for live cells, single cells, and fraction of GFP-negative cells.

Results: The graph in FIG. 72 shows the results of flow cytometry analysis of Cas-mediated editing at the RHO locus in APRE19 RHO-GFP cells 14 days post-transfection. Eighteen different spacers (indicated by the individual data points) targeting the RHO Exon 1 locus were used for each of the different CasX variants (438, 488, and 491) used in this experiment. Each data point is an average measurement of 3 replicates for an individual spacer. The median values for the constructs were: 438 (48.4); 488 (59.0) and 491 (56.4), indicating that under the conditions of the assay, each of the CasX variants with appropriate guides were able to specifically edit in APRE19 RHO-GFP reporter cells at a high level while the construct with a non-targeting spacer resulted in no editing (data not shown).

TABLE 25 Guide encoding sequences SPACER SEQUENCE 174 GUIDE + SPACER SEQUENCE Spacer (SEQ ID NO) (SEQ ID NO) 11.13 3628 3646 11.14 3629 3647 11.15 3630 3648 11.16 3631 3649 11.17 3632 3650 11.18 3633 3651 11.19 3634 3657 11.20 3635 3653 11.21 3636 3654 11.22 3637 3655 11.23 3638 3656 11.24 3639 3657 11.25 3640 3658 11.26 3641 3659 11.27 3642 3660 11.28 3643 3661 11.29 3644 3662 11.1  3645 3663

Example 27. Design of Improved Guides Based on Predicted Secondary Structure Stability Methods

A computational method was employed to predict the relative stability of the ‘target’ secondary structure, compared to alternative, non-functional secondary structures. First, the ‘target’ secondary structure of the gRNA was determined by extracting base-pairs formed within the RNA in the CryoEM structure for CasX 1.1. For prediction of RNA secondary structure, the program RNAfold was used (version 2.4.14). The ‘target’ secondary structure was converted to a ‘constraint string’ that enforces bases to be paired with other bases, or to be unpaired. Because the triplex is unable to be modeled in RNAfold, the bases involved in the triplex are required to be unpaired in the constraint string, whereas all bases within other stems (pseudoknot, scaffold, and extended stems) were required to be appropriately paired. For guide scaffolds 2 (SEQ ID NO:5), 174 (SEQ ID NO:2238), and 175 (SEQ ID NO:2239), this constraint string was constructed based on sequence alignment between the scaffold and scaffold 1 (SEQ ID NO:4) outside of the extended stem, which can have minimal sequence identity. Within the extended stem, bases were assumed to be paired according to the predicted secondary structure for the isolated extended stem sequence. See Table 26 for a subset of sequences and their constraint strings.

TABLE 26 Constraint strings to represent the ‘target secondary structure’ in RNAfold algorithm. Name Constraint string Scaffold 1 (w/5′ truncation (((((.xxx..........xxxxx))))).((.((((((((...))))).)))))...(((((((((((((((.......))))))))))).))))..xxxxx as in CryoEM structure) Scaffold 2 ....(((((.xxx.........xxxxx.)))))....((((((((...))))).))).....((.(((((((((((((......)))))))))))))..))..xxxxx Scaffold 174 ...(((((.xxx.........xxxxx.)))))....((((((((...)))))..))).....((((((((.....))))))))..xxxxx Scaffold 175 ...(((((.xxx.........xxxxx.)))))....((((((((...))))).))).....((.(((((((((....)))))))))..))..xxxxx

Secondary structure stability of the ensemble of structures that satisfy the constraint was obtained, using the command: ‘RNAfold -p0 --noPS -C’ And taking the ‘free energy of ensemble’ in kcal/mol (AG constraint). The prediction was repeated without the constraint to get the secondary structure stability of the entire ensemble that includes both the target and alternative structures, using the command: ‘RNAfold -p0 -noPS’ and taking the ‘free energy of ensemble’ in kcal/mol (ΔG_all).

The relative stability of the target structure to alternate structures was quantified as the difference between these two AG values: ΔΔG=ΔG_constraint−ΔG_all. A sequence with a large value for ΔΔG is predicted to have many competing alternate secondary structures that would make it difficult for the RNA to fold into the target binding-competent structure. A sequence with a low value for ΔΔG is predicted to be more optimal in terms of its ability to fold into a binding-competent secondary structure.

Results

A series of new scaffolds was designed to improve scaffold activity based on existing data and new hypotheses. Each new scaffold comprised a set of mutations that, in combination, were predicted to enable higher activity of dsDNA cleavage. These mutations fell into the following categories: First, mutations in the 5′ unstructured region of the scaffold were predicted to increase transcription efficiency or otherwise improve activity of the scaffold. Most commonly, scaffolds had the 5′ “GU” nucleotides deleted (scaffolds 181-220: SEQ ID NOS: 2242-2280). The “U” is the first nucleotide (U1) in the reference sequence SEQ ID NO:5. The G was prepended to increase transcription efficiency by U6 polymerase. However, removal of these two nucleotides was shown, surprisingly, to increase activity (FIG. 66). Additional mutations at the 5′ end include (a) combining the GU deletion with A2G, such that the first transcribed base is the G at position 2 in the reference scaffold (scaffold 199: SEQ ID NO:2259): (b) deleting only U1 and keeping the prepended G (scaffold 200: SEQ ID NO:2260); and (c) deleting the U at position 4, which is predicted to be unstructured and was found to be beneficial when added to scaffold 2 in a high-throughput CRISPRi assay (scaffold 208: SEQ ID NO:2268).

A second class of mutations was to the extended stem region. The sequence for this region was chosen from three possible options: (a) a “truncated stem loop” which has a shorter loop sequence than the reference sequence extended stem (the scaffolds 64 and 175 contain this extended stem: SEQ ID NOS: 2106 and 2239, respectively) (b) Uvsx hairpin with additional loop-distal mutations [A99] and G65T to fully base-pair the extended stem (the scaffold 174: SEQ ID NO: 2238) contains this extended stem); or (c) an “MS2(U15C)” hairpin with the same additional loop-distal mutations [A99] and G65T as in (b). These three extended stems classes were present in scaffolds with high activity (e.g. see FIG. 65), and their sequences can be found in Table 27.

TABLE 27 Sequences of extended stern regions used in novel scaffolds. Incorporated in Scaffolds Extended stem name Extended stem sequenee (SEQ ID NO) truncated stein loop GCGCUUACGGACUUCGGUCCGUAAGAAGC 2239, 2242-2244, 2246, 2255-2258 (SEQ ID NO: 4291) UvsX, -99 G65T GCUCCCUCUUCGGAGGGAGC (SEQ ID 2238, 2245, 2250-2254, 2259-2280 NO: 4292) MS2(U15C), -99 G65T GCUCACAUGAGGAUCACCCAUGUGAGC 2249 (SEQ ID NO: 4293)

Thirdly, a set of mutations was designed to the triplex loop region. This region was not resolved in the CryoEM structure of CasX 1.1, likely because it does not form base-pairs and thus is more flexible. This region tolerates mutations, with certain mutations having beneficial effects on RNP binding, based on CRISPRi data from scaffold 2 (FIG. 63). The C18G substitution within the triplex loop was already incorporated in the scaffold 174. The following mutations were added to scaffold 174, that were not immediately adjacent to the C18G substitution in order to limit potential negative epistasis between these mutations: {circumflex over ( )}U15 (insertion of U before nucleotide 15 in scaffold 2), {circumflex over ( )}U17, and C16A (scaffolds 208, 210, and 209: SEQ ID NOS: 2268, 2270, 2269, respectively).

Fourth, a set of mutations was designed to systematically stabilize the target secondary structure for the scaffold. For background, RNA polymers fold into complex three-dimensional structures that enforce their function. In the CasX RNP, the RNA scaffold forms a structure comprising secondary structure elements such as the pseudoknot stem, a triplex, a scaffold stem-loop, and an extended stem-loop, as evident in the Cryo-EM characterization of the CasX RNP 1.1. These structural elements likely help enforce a three dimensional structure that is competent to bind the CasX protein, and in turn enable conformational transitions necessary for enzymatic function of the RNP. However, an RNA sequence can fold into alternate secondary structures that compete with the formation of the target secondary structure. The propensity of a given sequence to fold into the target versus alternate secondary structures was quantified using computational prediction, similar to the method described in (Jarmoskaite, I., et al. 2019. A quantitative and predictive model for RNA binding by human pumilio proteins. Molecular Cell 74(5), pp. 966-981.e18) for correcting observed binding equilibrium constants for a distinct protein-RNA interaction, and using RNAfold (Lorenz, R., Bernhart, S. H., Höner Zu Siederdissen, C., et al. 2011. ViennaRNA Package 2.0. Algorithms for Molecular Biology 6, p. 26) to predict secondary structure stability (see Methods).

A series of mutations were chosen that were predicted to help stabilize the target secondary structure, in the following regions: The pseudoknot is a base-paired stem that forms between the 5′ sequence of the scaffold and sequence 3′ of the triplex and triplex loop. This stem is predicted to comprise 5 base-pairs, 4 of which are canonical Watson-Crick pairs and the fifth is a noncanonical G:A wobble pair. Converting this G:A wobble to a Watson Crick pair is predicted to stabilize alternative secondary structures relative to the target secondary structure (high ΔΔG between target and alternative secondary structure stabilities; Methods). This aberrant stability comes from a set of secondary structures in which the triplex bases are aberrantly paired. However, converting the G to an A or a C (for an A:A wobble or C:A wobble) was predicted to lower the ΔΔG value (G8C or G8A added to scaffolds 174 and 175+C18G). A second set of mutations was in the triplex loop: including a U15C mutation and a C18G mutation (for scaffold 175 that does not already contain this variant). Finally, the linker between the pseudoknot stem and the scaffold stem was mutated at position 35 (U35A), which was again predicted to stabilize the target secondary structure relative to alternatives.

Scaffolds 189-198 (SEQ ID NOS:2250-2258) included these predicted mutations on top of scaffolds 174 or 175, individually and in combination. The predicted change in ΔΔG for each of these scaffolds is given in Table 28 below. This algorithm predicts a much stronger effect on ΔΔG with combining multiple of these mutations into a single scaffold.

TABLE 28 Predicted effect on target secondary structure stability of incorporating specific mutations individually or in combination to scaffolds 174 or 175. Starting Scaffold ΔΔG Effect of mutation(s) ΔΔG_mut- scaffold Mutation(s) (kcal/mol) ΔΔG_starting_scaffold (kcal/mol) 174 — 0.17 — 174 G8A −0.74 −0.91 174 G8C −0.32 −0.49 174 U15C −0.02 −0.19 174 U35A −0.22 −0.39 174 G8A, U15C, −1.34 −1.51 U35A 175 — 3.23 — 175 G8A 3.15 −0.08 175 G8C 3.15 −0.08 175 U35A 3.07 −0.16 175 U15C 0.78 −2.45 175 C18G 0.43 −2.80 175 G8A, T15C, −1.03 −4.26 C18G, T35A

A fifth set of mutations was designed to test whether the triplex bases could be replaced by an alternate set of three nucleotides that are still able to form triplex pairs (Scaffolds 212-220: SEQ ID NOS:2272-2280). A subset of these substitutions are predicted to prevent formation of alternate secondary structures.

A sixth set of mutations were designed to change the pseudoknot-triplex boundary nucleotides, which are predicted to have competing effects on transcription efficiency and triplex formation. These include scaffolds 201-206 (SEQ ID NOS:2261-2266).

Example 28: In Vitro Cleavage Assays with NTC PAMs

In vitro cleavage assays were performed essentially as described in Example 19, using CasX 2 (SED ID NO:2). CasX 119, and CasX 438 complexed with single guide 174 with spacer 7.37 targeted against B2M. Fluorescently labeled dsDNA targets that would be complementary with a 7.37 spacer and either a TTC, CTC, GTC, or ATC PAM were used (The DNA sequences used to generate each dsDNA substrate are shown in Table 29. The PAM sequences for each are bolded. TS—target strand. NTS—Non-target strand). Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. The monophasic fit of the combined replicates is shown. During the assay, samples were taken at at 0.25, 0.5, 1, 2, 5, 10, 30, and 60 minutes. Gels were imaged with an Amersham Typhoon and quantified using the IQTL 8.2 software. Apparent first-order rate constants for non-target strand cleavage (k_(cleave)) were determined for each Casx:sgRNA complex on each target. Rate constants for targets with non-TTC PAM were compared to the TTC PAM target to determine whether the relative preference for each PAM was altered for a given CasX variant. The results are shown in FIG. 73 (the monophasic fit of the combined replicates is shown) and Table 30. For all Cas X variants, the TTC PAM target sequence supported the highest cleavage rate, followed by the ATC, then the CTC, and finally the GTC target sequence. The CTC target supported cleavage 3.5-4.3% as fast as the TTC target; the GTC target supported cleavage 1.0-1.4% as fast; and the ATC target supported cleavage 6.5-8.3% as fast. Despite the lower k_(cleave) rates for the non-TTC PAM, the cleavage rates of the variants allow targets with ATC or CTC PAMs to be cleaved nearly completely within 10 minutes, and these increased cleavage rates relative to the wild-type CasX may be sufficient for effective genome editing in a human cell, supporting the utility of the CasX variants having an increased ability to utilize a larger spectrum of PAM sequences.

TABLE 29  Sequences of DNA substrates used in in vitro PAM cleavage assay. Assay Combination DNA Substrate Sequence* 7.37 TTC AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAATGCTGTCAGCTTCA (SEQ PAM TS ID NO: 4404) 7.37 TTC TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCT (SEQ PAM NTS ID NO: 4405) 7.37 CTC AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAGTGCTGTCAGCTTCA (SEQ PAM TS ID NO: 4406) 7.37 CTC TGAAGCTGACAGCACTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCT (SEQ PAM NTS ID NO: 4407 7.37 GTC AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGACTGCTGTCAGCTTCA(SEQ PAM TS ID NO: 4408) 7.37 GTC TGAAGCTGAGCAGTCGGGCCGAGATGTTCGCTCCGTGGCCTTAGCTGTGCTCGCGCT (SEQ PAM NTS ID NO: 4409) 7.37 ATC AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGATTGCTGTCAGCTTCA (SEQ PAM TS ID NO: 4410) 7.37 ATC TGAAGCTGACAGCAATCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCT (SEQ PAM NTS ID NO: 4411) *PAM indicated in bold

TABLE 30 Cleavage Rates k_(cleave) Rate* CasX TTC CTC GTC ATC 2 0.267 min⁻¹ 9.29E−3 min⁻¹ 3.75E−3 min⁻¹ 1.87E−2 min⁻¹ (0.035) (0.014) (0.070) 119  8.33 min⁻¹ 0.303 min⁻¹ 8.64E−2 min⁻¹ 0.540 min⁻¹ (0.036) (0.010) (0.065) 438  4.94 min⁻¹ 0.212 min⁻¹ 1.31E−2 min⁻¹ 0.408 min⁻¹ (0.043) (0.013) (0.083) *For all non-NTC PAMs, the relative cleavage rate as compared to the TTC rate for that variant is shown in parentheses. 

1-222. (canceled)
 223. An engineered CRISPR protein, wherein the engineered protein comprises amino acids 648-812 of a RuvC DNA cleavage domain of SEQ ID NO: 2, comprising one or more amino acid modifications thereto.
 224. The engineered CRISPR protein of claim 223, wherein the amino acid modification is a modification at one or more amino acid positions selected from the group consisting of I658, A708, and P793.
 225. The engineered CRISPR protein of claim 223, wherein the amino acid modification is an amino acid substitution.
 226. The engineered CRISPR protein of claim 225, wherein the substitution is a substitution at amino acid position A708 or is a substitution at amino acid position I658.
 227. The engineered CRISPR protein of claim 226, wherein the substitution is A708K.
 228. The engineered CRISPR protein of claim 226, wherein the substitution is I658V.
 229. The engineered CRISPR protein of claim 223, wherein the amino acid modification is an amino acid deletion.
 230. The engineered CRISPR protein of claim of claim 229, wherein the deletion is at amino acid position P793.
 231. The engineered CRISPR protein of claim 223, further comprising amino acids 922-978 of a RuvC DNA cleavage domain of SEQ ID NO: 2, or at least 70% sequence identity thereto.
 232. The engineered CRISPR protein of claim 231, further comprising one or more of: (a) a non-target strand binding (NTSB) domain; (b) a target strand loading (TSL) domain; (c) a helical I domain; (d) a helical II domain; and (e) an oligonucleotide binding domain (OBD).
 233. The engineered CRISPR protein of claim 232 comprising a NTSB domain, wherein the NTSB domain comprises amino acids 101-191 of SEQ ID NO: 1, or at least 70% sequence identity thereto.
 234. The engineered CRISPR protein of claim 233 comprising a TSL domain, wherein the TSL domain comprises amino acids 813-921 of SEQ ID NO: 2, or at least 70% sequence identity thereto.
 235. The engineered CRISPR protein of claim 234 comprising a helical I domain, wherein the helical I domain is a chimeric helical I domain derived from the helical I domain sequences of SEQ ID NO: 1 and SEQ ID NO:
 2. 236. The engineered CRISPR protein of claim 235, wherein the chimeric helical I domain comprises amino acids 59-102 of SEQ ID NO: 2, or at least 70% sequence identity thereto, and comprises amino acids 192-332 of SEQ ID NO: 1, or at least 70% sequence identity thereto.
 237. The engineered CRISPR protein of claim 236 comprising a helical II domain, wherein the helical II domain comprises amino acids 334-501 of SEQ ID NO: 2, or at least 70% sequence identity thereto.
 238. The engineered CRISPR protein of claim 237, wherein the helical II domain comprises a substitution at amino acid position L379 of SEQ ID NO:
 2. 239. The engineered CRISPR protein of claim 238, wherein the substitution is L379R.
 240. The engineered CRISPR protein of claim 237, wherein the helical II domain comprises a substitution at amino acid position F399 of SEQ ID NO:
 2. 241. The engineered CRISPR protein of claim 240, wherein the substitution is F399L.
 242. The engineered CRISPR protein of claim 237 comprising an OBD, wherein the OBD comprises amino acids 1-58 of SEQ ID NO: 2, or at least 70% sequence identity thereto, and comprises amino acids 502-647 of SEQ ID NO: 2, or at least 70% sequence identity thereto.
 243. The engineered CRISPR protein of claim 223 comprising: (a) a non-target strand binding (NTSB) domain; (b) a target strand loading (TSL) domain; (c) a chimeric helical I domain; (d) a helical II domain; (e) an oligonucleotide binding domain (OBD); and (f) a RuvC DNA cleavage domain, wherein the engineered CRISPR protein is a chimeric protein, wherein the NTSB domain is derived from SEQ ID NO: 1, and wherein the TSL, helical II, OBD, and RuvC DNA cleavage domains are derived from SEQ ID NO:
 2. 244. The engineered CRISPR protein of claim 243, comprising: (a) a RuvC DNA cleavage domain comprising a substitution at amino acid position I658 of SEQ ID NO: 2; (b) a RuvC DNA cleavage domain comprising a substitution at amino acid position A708 of SEQ ID NO: 2; (c) a helical II domain comprising a substitution at amino acid position L379 of SEQ ID NO: 2; and/or (d) a helical II domain comprising a substitution at amino acid position F399 of SEQ ID NO:
 2. 245. The engineered CRISPR protein of claim 244, wherein the chimeric helical I domain comprises amino acids 59-102 of SEQ ID NO: 2, or at least 70% sequence identity thereto, and comprises amino acids 192-332 of SEQ ID NO: 1, or at least 70% sequence identity thereto.
 246. The engineered CRISPR protein of claim 245, comprising: (a) a RuvC DNA cleavage domain comprising the amino acid substitution I658V; (b) a RuvC DNA cleavage domain comprising the amino acid substitution A708K; (c) a RuvC DNA cleavage domain comprising a deletion at amino acid position P793; (d) a helical II domain comprising the amino acid substitution L379R; and (e) a helical II domain comprising the amino acid substitution F399L.
 247. The engineered CRISPR protein of claim 243, wherein the engineered CRISPR protein exhibits one or more improved characteristic as compared to SEQ ID NO:
 2. 248. The engineered CRISPR protein of claim 247, wherein the one or more improved characteristic is selected from the group consisting of: improved folding; improved binding affinity to a guide nucleic acid (gNA); improved binding affinity to a target DNA; improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA; improved unwinding of the target DNA; increased editing activity; improved editing efficiency; improved editing specificity; increased nuclease activity; increased target strand loading for double strand cleavage; decreased target strand loading for single strand nicking; decreased off-target cleavage; improved binding of non-target DNA strand; improved protein stability; improved protein solubility; improved protein:gNA complex (RNP) stability; improved protein:gNA complex solubility; improved protein yield; improved protein expression; and improved fusion characteristics.
 249. The engineered CRISPR protein of claim 223, wherein the protein is selected from the group consisting of SEQ ID NO: 3505, SEQ ID NO: 3506, SEQ ID NO: 3507, and SEQ ID NO: 3548, or a protein comprising at least 70% sequence identity thereto.
 250. A variant of a reference guide nucleic acid scaffold (gNA variant) capable of binding an engineered CRISPR protein comprising amino acids 648-812 of a RuvC DNA cleavage domain of SEQ ID NO: 2 comprising one or more amino acid modifications thereto, and wherein: (a) the gNA variant comprises at least one modification compared to the reference guide nucleic acid scaffold sequence; and (b) the gNA variant exhibits one or more improved characteristics compared to the reference guide nucleic acid scaffold.
 251. A gene editing pair comprising: (a) an engineered CRISPR protein comprising amino acids 648-812 of a RuvC DNA cleavage domain of SEQ ID NO: 2 comprising one or more amino acid modifications thereto; and (b) a first guide nucleic acid.
 252. A method of editing or modifying a target DNA, comprising contacting the target DNA an engineered CRISPR protein comprising amino acids 648-812 of a RuvC DNA cleavage domain of SEQ ID NO: 2 comprising one or more amino acid modifications thereto, and a first guide nucleic acid, wherein the contacting results in editing or modification of the target DNA. 